Chromosomal fragmentations or damage in sperm DNA has considerable value in determination of semen quality. However, rapid and/or simple method to assess sperm DNA integrity in buffalo has apparently not been reported. In the present study, SCD was used for the first time in buffalo bulls for assessment of sperm DNA fragmentation. A modified SCD protocol, under bright field microscope was developed and validated by comparison with other routine tests which can be used for processing of samples. The DNA fragmentation index (DFI) from SCD was correlated with semen quality parameters viz. viability (r = −0.68, p < 0.05), membrane integrity (r = −0.74, p < 0.05) and capacitation status (r = −0.69, p < 0.05). The amount of DNA fragmentation assessed by SCD was highly correlated (R = 0.874, p < 0.05) with results of acridine orange test (AOT), a traditional method of assessing DNA damage. There were no significant differences between two observers with regards to scoring dispersion patterns. Therefore, the SCD test can be routinely used for detection of DNA fragmentation in buffalo sperm, with potential for replacing conventional time consuming tests.