Telling the difference between a male and a female kitten can be tricky, depending on the age of the cat. Kittens younger than 8 weeks old have yet to develop their genitalia, making the difference between the genders harder to distinguish. After 8 weeks, a kitten's genitalia should develop to the point where the differences between the genders are more apparent.
Pick up the kitten.
Turn the kitten so that you can see its hindquarters.
Lift the kitten's tail. The kitten's anus is directly beneath the tail and its genitalia is beneath the anus. If the kitten is male, its genitalia resembles a round dot or period. If the kitten is female, its genitalia resembles a vertical line.
A kitten's coat colour and markings may indicate its gender. For genetic reasons, nearly all calico and tortoishell cats are female.