The genitals of kittens are not as developed as in adult cats, so sex determination in kittens becomes a very challenging task. Looking at them for the first time, even the professionals find it tricky to distinguish a female kitten from a male. Yet there are a few characteristic differences that can be observed in kittens as young as five weeks old like genital spacing, coat color, and behavior indicating their sex.
Careful Handling

Don’t disturb the mother and kitten within the first few days of their life. If a kitten is handled too much during this time, the mother might abandon it and stop nursing it altogether. So don’t be very anxious to determine the sex of your kittens. Instead, wait until at least two weeks or longer before you start looking after the kittens.
While trying to sex determination in kittens, it is necessary to not stress out at all. Start with picking him up, gently petting him, and snuggling him close to your body. Scratching his/her back right at the base of the tail may make him/her ease their body and sometimes automatically lift it, giving you a bird’s eye view of the parts in question. But if a kitten is irritated or panicky, then leave the inspection process to a later date or time.
Identifying Genitals

At about two weeks old, some breeders link the appearance of kitten genitals to punctuation marks. However, the female kitten’s genitals will resemble an upside-down exclamation mark, with the vertical vaginal slit sitting just below the anus and with very less spacing in between. A male kitten’s organ looks more like a colon, with the penis placed just below the anus and separated by enough space to accommodate his testicles. You might even be able to see small lumps in that space, which will grow as the kitten grows older.
Coat Color

Yet another simple, but somewhat reliable, way to tell the sex of your kitten is by the color of its coat. Cats with three colors or more are mostly always female. The chromosomal makeup of female cats is quite responsible for this type of coloration, so you can pretty much guarantee any cat is a female with this type of color combination. Orange-colored or ginger cats, however, are most often male. However, this trait is not as reliable as the tri-colored trait, as only three-quarters of entire ginger-colored cats are male.
Behavior

As kittens grow up, they start to develop their unique personalities. Male kittens may start to roam around more as they approach sexual maturity. They may also become territorial, often wandering to establish their home base and ward off trespassers. Males are also more restless and aggressive in nature.
Though some people claim male cats are friendlier and more outgoing than females. But this difference is, by no means is universal and cat personalities are just as varied as human personalities. So choose your cat by the connection you make with them upon the first interaction and pay less attention to its gender.
Handling Kittens

Even though you’ll need to take a look under their tails, be sure not to lift them by the tail, as this can cause them spine injuries. It’s best to simply lift the tail while a kitten lays on a flat surface or hold them on its back while supporting its head and neck.
Finding the Differences
There are two main differences to keep an eye on when sex determination in kittens. The first is commonly known as the anogenital distance, which means the distance between the anus and the genitals and the other in the shape of the genitals.
Male Kittens:
In males, the penis is located at a greater distance from the anus, as a small round dot with the testicles present in between.
Female Kittens:
In females, the vulva is located just below the anus and looks like a vertical slit.
Determining the sex of a kitten can be quite challenging. Especially before about six to eight weeks of age. But with a bit of practice, you will be able to determine if your kitten is a ‘He’ or a ‘She.