Hybrid male sterility (HMS) is typically the first mechanism fortifying reproductive isolation resulting from genomic incompatibilities. Three interspecies feline breeds derived from domestic cat crosses to wild cat species (Asian leopard cat and African serval) manifest HMS through several generations of backcrossing before eventually regaining fertility. This work utilized 199 hybrid individuals with varying fertilities in a genome wide association study (GWAS) comprising 63,000 genome wide SNPs. Leveraging these results with whole-testis transcriptome sequencing and quantitative real-time PCR data facilitated the comparison of transcripts in sterile and fertile hybrids. This dissertation describes four loci with highly significant and fifty with moderately significant association to sterility within each individual hybrid domestic breed and combinations of breeds. These associations help identify epistatic targets for hybrid incompatibility contributing to sterility. Comparative QTL mapping between pairs of species provides a framework to describe the accumulation of clade-specific reproductive isolating loci. Detailed exploration of gene misregulation between domestic and hybrid individuals, as well as between littermate hybrids of varying fertilities outlines a pattern of expression consistent with a meiotic sex-chromosome inactivation failure in early generations and apoptotic failure in later hybrid generations. Combining comparative genomic association and transcriptomic characterization among hybrid felids of varying divergence, new insight is gained into the mechanisms of mammalian reproductive isolation.