Sexing ostrich chicks and juveniles before the dimorphous appearance of the plumage that starts at 14 months of age is proving difficult and cumbersome for Botswana farmers. This problem delays early selection of birds for breeding and trading. It also means that birds are often sold as a mixture of males and females. DNA-based sex identification provides a solution and is amenable to large-scale application. The application of the multiplex polymerase chain reaction (PCR) was used to determine the sex of 6-months old juvenile ostriches. The blood from four mature males and four mature females were used to verify the assay. The test group consisted of 19 randomly selected six-month old birds, which turned out to be 10 males and nine females. The PCR technique uses two pairs of primers. The primers SS1 and SS2 amplify a 650 base pair (bp) female-specific fragment, while the primers L014a and L014b amplify a fragment about 280 bp, from both sexes. The females were thus distinguished by amplification of two bands after agarose gel electrophoresis, and the males by one band. Following the successful application of this technique using DNA extracted from blood, further investigations have to be conducted, using DNA extracted from other body tissue samples such as feathers or skin. A scenario is envisaged whereby, during tagging of the chicks, a body tissue sample such as a blood feather could be sent to the laboratory for DNA sexing. This technique would aid farmers in identifying the sex of their birds at a young age.