Chromosome heteromorphism can lead to non-random segregation at meiosis (Wang et al., 2010). A common example of autosomal hetermorphism is observed in chromosomes carrying the Nucleolar Organiser Region (NOR), which is a region of tandemly repeated rDNA clusters. Differences in the number of clusters, different transcriptional activity and DNA methylation levels between homologous NORs lead to size heteromorphisms. In the platypus, chromosome 6 contains a large heteromorphic NOR. Interestingly, this chromosome is homologous to the therian X-chromosome. Therefore, we were very interested in investigating whether this size difference may lead to non-random segregation of chromosome 6. Firstly, we observed that the heteromorphism exists in both sexes being more prominent in females. 5-Aza-dC treatment showed that chromosome 6 hypermethylation contributes to the heteromorphism in females but not in males and silver staining indicated that both NORs are active but revealed a gap in the males NOR. This showed that sex specific differences exist between the NORs on the chromosome 6 homologs. In our segregation studies on male meiosis we found that the chromosome 6 carrying the short NOR segregates preferentially with X-bearing sperm whereas Y-bearing sperm carries the short and long chromosome 6 in equal proportions. Negative selection maybe acting against the X-bearing sperm that carries the long chromosome 6. Supporting this idea, we have observed consistently lower counts of X-bearing sperm than Y-bearing sperm in three male individuals. Together this study provides the first evidence of non-random segregation of an autosome in the platypus as well as sex-specific NOR differences. The mechanisms behind this segregation bias as well as its exact link with the observed NOR differences remain to be investigated.