Establishing the gender of a cat is not an easy thing to do. In fact, many breeders will freely admit that they make a mistake from time to time. The difficulty arises because cats' genital areas look remarkably similar whether the cat is male or female. There is no convenient obvious difference in colouring or appearance. Unlike dogs, a tomcat's penis are not distended. Both female and male cats have nipples. For this reason many breeders will only sex their cats after 8 weeks when anatomical differences have become more obvious, but some breeders reckon that kittens just after birth are easiest to sort.
So how does one differentiate between a male and female kitten? The diagram below shows how the genital area differs between the sexes. There are two major differences to look for: 1. the shape of the vulva, more elongated with a definite slit, vs penis (round); 2. the distance from the anus. In male kittens, the distance between the anus and the penis is larger to allow the space for the scrotum.
To determine the sex of a kitten, put the cat on the table and lift up the tail. The genital areas are very close to the base of the tail with the anus immediately under the tail. In the male kitten, the scrotum is located below the anus and above the back of the hind legs. The penis is immediately below the scrotum, and it is not visible. In females the vulva is located below the anus and above the back legs. If you have a number of mixed (male/female) kittens compare the kittens side by side. That way the differences will be much easier to spot.
With older cats, you can regard it as definitive proof that you have a female on your hands when she presents you with a litter of kittens, but most owners would prefer not to wait that long!