Authors' Contribution AN and MJ conceived the idea and executed the project. RBK carried out the genomic work. MZS and MEB assisted in data analysis and interpretation. RBK and MJ assisted in manuscript write up and editing. Key words β-casein, Cholistani cattle, Variants β-casein is second most abundant protein of cow's milk. β-casein gene is highly polymorphic. A1 and A2 are the frequently occurred variants. A1 is recognized as potential cause of several human diseases. It is important to evaluate the A1/A2 β-casein status in milk. Current study was conducted to molecular characterize the exonic regions of β-casein gene and to explore the status of A1/A2 β-casein type in Cholistani cattle breed of Pakistan. Blood samples of Cholistani Cattle were collected from Government Livestock Farm, Jugait Peer, Bahawalpur. Genomic DNA was extracted from whole blood by organic method. PCR primers were designed and optimized according to respective melting temperatures. PRALINE tool, MEGA 6.0 and POPGENE tool were utilized for phylogenetic tree construction and statistical analysis, respectively. Characterization of physical and chemical properties of β-casein protein was performed by ProtParam and SWISS MODEL was utilized for protein model prediction. Sequencing results of amplified PCR products revealed total 9 SNPs including 1 in exonic and 8 in intronic regions of β-casein gene. Results represent the presence of A2 allele in milk of Cholistani Cattle of Pakistan. Multiple sequence alignment represented the presence of MKVLILACLVALALARE and QRAVPVQALLLNQE as highly conserved regions. Phylogenetic analysis revealed the evolutionary relationship among Cholistani cattle of Pakistan, Bos indicus and Bos taurus. β-casein gene is highly polymorphic and A2 allele is present in Cholistani Cattle of Pakistan.