This study investigates in silico the genetic diversity of CSN2 on its evolutionary and differentiation within and among species and also examines the attendant effects of polymorphism on the functionality of CSN2. A total of 22 CNS2 gene sequences with corresponding amino acids belonging to 8 species [cattle (3), buffalo (3), camel (3), goat (3), horse (1), rabbit (3), rat (3) and mice (3) were retrieved from Genbank (www.ncbi.nlm.nih.gov).  All sequences were trimmed to equal length (501bp) corresponding to the same region. Sequences alignment, translation and comparison were done with ClustalW using IUB substitution matrix, gap open penalty of 15 and gap extension penalty of 6.66. The alignment revealed high polymorphism of CNS2 sequences within and among species. The Dxy inferred using p-distance revealed a maximum value of 0.68 between horse and goat and a minimum value of 0.03 between cattle and buffalo. The hypothesis of strict neutral evolution was rejected for both genes as P<0.05 for species. Also allelic sequence evolution was entirely driven by positive selection. In silico functional analysis of non-synonymous mutations using PANTHER revealed that, for CSN2, 11 of the amino acid substitutions in the peptide binding region (cattle 5, goat 3 and sheep 1) did not impair protein function. However, 3 substitutions in cattle were predicted to be harmful to protein function, also 2 potentially deleterious SNPs; 1 (Glu36Lys) for cattle and 1 (Leu206Pro) for horse were found. The NJ phylogeny revealed trans-species evolution however, UPGMA tree topology was species-wise. In conclusion, all identified deleterious SNPs should be taken into account while selecting a stock for milk production.