Abstract Methods to determine the sex from tissue samples of mammals include the amplification of Y chromosome specific regions, which should only amplify from males, or amplification of homologous regions of the X and Y chromosome containing XY specific SNPs. A disadvantage of the first approach is that PCR failure can be misinterpreted as the identification of a female. The latter approach is proposed to identify PCR failure through non-amplification of the X homologue, which should be present in both sexes. This method is therefore potentially more suitable for molecular sexing of degraded DNA with a high probability of PCR failure, such as for example, ancient DNA samples. Here, we investigate the validity of this assumption regarding the use of XY homologue PCR assays for molecular sexing of ancient DNA. We tested a primer set targeting the ZFX/ZFY alleles using ancient DNA extracts from 100 to 4500 years old bowhead whale samples, and for comparison on dilution series from modern bowhead whales of known sex. DNA sequencing of PCR products obtained from the ancient material confirmed a higher proportion of successful PCR amplifications of the X homologue over the Y homologue. This potentially biased sex determination was further assessed by testing highly diluted DNA extracts of modern samples, for which a consistently higher success rate of PCR amplification and lower PCR cycle threshold was found for the X homologue from females than either homologue from males. This is most likely due to the higher copy number of the X homologue in females, although other yet unknown attributes of the protocol may also cause the observed bias. The current case study provides a valuable example of a potential pitfall in molecular sex determination of ancient mammal DNA in zooarchaeology. High-throughput sequencing methods, in which sufficiently large numbers of reads can be unambiguously mapped to X and Y regions, should overcome such biases and be the most robust approach for molecular sex determination using degraded DNA.