Abstract Background Recent progress in exploring the Y-chromosome gene content in humans, mice and cats have suggested that "autosome-to-Y" transposition of the male fertility genes is a recurrent theme during the mammalian Y-chromosome evolution. These transpositions are lineage-dependent. The purpose of this study is to investigate the lineage-specific Y-chromosome genes in bovid. Results We took a direct testis cDNA selection strategy and discovered two novel gene families, ZNF280BY and ZNF280AY, on the bovine (Bos taurus) Y-chromosome (BTAY), which originated from the transposition of a gene block on the bovine chromosome 17 (BTA17) and subsequently amplified. Approximately 130 active ZNF280BY loci (and ~240 pseudogenes) and ~130 pseudogenized ZNF280AY copies are present over the majority of the male-specific region (MSY). Phylogenetic analysis indicated that both gene families fit with the "birth-and-death" model of evolution. The active ZNF280BY loci share high sequence similarity and comprise three major genomic structures, resulted from insertions/deletions (indels). Assembly of a 1.2 Mb BTAY sequence in the MSY ampliconic region demonstrated that ZNF280BY and ZNF280AY, together with HSFY and TSPY families, constitute the major elements within the repeat units. The ZNF280BY gene family was found to express in different developmental stages of testis with sense RNA detected in all cell types of the seminiferous tubules while the antisense RNA detected only in the spermatids. Deep sequencing of the selected cDNAs revealed that different loci of ZNF280BY were differentially expressed up to 60-fold. Interestingly, different copies of the ZNF280AY pseudogenes were also found to differentially express up to 10-fold. However, expression level of the ZNF280AY pseudogenes was almost 6-fold lower than that of the ZNF280BY genes. ZNF280BY and ZNF280AY gene families are present in bovid, but absent in other mammalian lineages. Conclusions ZNF280BY and ZNF280AY are lineage-specific, multi-copy Y-gene families specific to Bovidae, and are derived from the transposition of an autosomal gene block. The temporal and spatial expression patterns of ZNF280BYs in testis suggest a role in spermatogenesis. This study offers insights into the genomic organization of the bovine MSY and gene regulation in spermatogenesis, and provides a model for studying evolution of multi-copy gene families in mammals.