A combination of chromosomal banding and fluorescence in situ hybridization (FISH) was used to characterize the karyotype of Boselaphus tragocamelus (nilgai) relative to the domestic cattle standard karyotype. G-, Q- and C-band karyotypes of nilgai are presented, and the chromosomal complement of nilgai is determined to be 2n = 46 (female FN = 60, male FN = 59; NAA = 56), consistent with previous reports for the species. Comparisons with cattle identified extensive monobrachial homologies with some noteworthy exceptions. Chromosome 25 is centrically fused to 24, and chromosome 16 is acrocentric. Both appear to have additional pericentromeric material not seen in the equivalent cattle acrocentrics. This pericentromeric chromatin may be the result of de novo additions or translocation of pericentromeric material from chromosome 6, which is shown to be centrically fused to 13 but is only about two-thirds the length of cattle 6. Comparisons with cattle demonstrated that nilgai chromosome 17 has undergone a paracentric inversion and that chromosome 20 has two blocks of interstitial constitutive heterochromatin. The identities of both chromosomes were confirmed by chromosomal FISH. Furthermore, chromosomal banding and FISH were used to determine that autosome 14 has been fused to the ancestral X and Y of nilgai to form compound neo-X and -Y chromosomes. Additional FISH analyses were conducted to confirm other proposed chromosome homologies and to identify nucleolar organizing regions within the nilgai complement.