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Spermatozoa in different species are morphologically and physiologically distinct, as well as under different post-copulatory selection regimes. Here we used published data on sperm cell proteomics and microarray expression profiles of the testis and sequential spermatogenic stages to elucidate trends in the evolution of male-related genes. A comparative proteomic analysis of the Mus musculus and Drosophila melanogaster sperm proteomes demonstrated the conservation of the functional composition of sperm proteomes. Despite this similarity, spermatozoa are known to be a rapidly evolving, highly species specific, cell type. One possible hypothesis that may explain the dichotomy of rapid evolution and conservative constraint is gene duplication. Consistently, a survey of retrotransposition, a RNA-based form of gene duplication, in mammals and D. melanogaster revealed that ~20% of known retrogenes encode novel sperm proteins. Further analysis demonstrated that retrotransposition has an important role in the generation of novel sperm genes that function in metabolism. Of particular interest was the observation that sperm retrogenes in mammals and Drosophila were enriched with functions in disparate metabolic pathways, which mirrored the different pathways underlying processes subject to post-copulatory sexual selection due to their role in sperm competition. In addition to this important role in the evolution of sperm there is a general, documented, trend that retrogene are expressed in the testis. However, little is known about the selection on retrogenes as they initially acquire their ability to be expressed. One hypothesis is that the region into which a retrogene is inserted has an especial influence on the evolution of its expression. To determine whether there is an observable effect of retrogene location on retrogene expression a new model for the identification of genomic regions enriched for genes expressed in the testis was developed. Utilising this method significant co-localisation of testisexpressed genes in the D. melanogaster genome was observed, together with a significant association between retrogene residence in these genomic regions and the acquisition of retrogene testis expression.