Pakistani river buffalo breeds (Azi-kheli, Kundi, Nili, Nili-Ravi and Ravi) are the least investigated ones for cytogenetic and genome studies. Although the first chromosomal study in buffalo was undertaken in 1960 however, there has been a long lapse until 2001. With the latest advancements in molecular biology tools and techniques including genome sequencing, PCR amplification and identification of SNPs of interest in different genes and high throughput microarray technology rapid progress in molecular determination of buffalo genome is expected in the near future. Similarly establishment of standard chromosome nomenclature of buffalo and clinical Cytogenetics has also been undertaken in Pakistan. Following paragraphs present the first ever review of chromosomal and genome studies at Cytogenetic and molecular genetic level providing a first comprehensive reference document for scientific use