At first glance, it is difficult to determine the sex of puppy, but you can easily figure it out through a quick visual inspection of the puppy's genitalia. Dogs are mammals and therefore have either male or female sex organs. When determining the sex of your puppies, try your best not to distress the puppies or their mother. Mother dogs are instinctually very protective of their young and can become easily upset if they are separated from their puppies.
Prepare to pick up your puppy by warming your hands; this is particularly important if the puppy is only a few days old. Gently pick up your puppy and hold it in your hands with its belly exposed, making sure that you are holding the puppy securely by supporting the its back.
Examine the puppy's genitalia. Located between the puppy's umbilical cord (or bellybutton) and anus. If the puppy has a penis, it is a male. (The penis will appear as a raised lump on the puppy's belly.) If the puppy has a vulva (vagina), it's a female. The vulva is located farther back then the penis and has a leaf-shaped appearance.
Gently place the puppy back with its mother.