This study investigated the genetic polymorphism of osteopontin (OPN) gene in Egyptian buffalo bulls in a trial for association with the quality of fresh semen. A total of 228 fresh semen ejaculates were collected from 57 buffalo bulls. Checking the fertility potential, the ejaculates were evaluated for volume, individual motility, live sperm, sperm abnormalities and concentration. The bulls' semen was grouped according to the individual motility into high (>60%, n=47) and low (<60%, n=10) quality. A fragment of 250 bp from intron 3 of OPN gene was amplified by PCR then genotyped by restriction fragment length polymorphism, single strand conformation polymorphism and DNA sequencing. The high-quality semen was significantly increased in individual motility % (p < 0.0001), live sperm % (p < 0.0001) and sperm concentration (p < 0.05) than the low quality. Also, the monomorphic pattern of OPN gene in both groups of bulls was noticed. However, five mutations were discovered including three nucleotide insertions (T 44, A49 and A62) and two nucleotide substitutions (G11>C and A108>G) when comparing the sequence with that of buffalo in the GenBank.  In conclusion, OPN gene might have no genetic variation and so it is not associated with semen quality in Egyptian buffalo bulls. Future studies with a larger number of populations on different regions of OPN gene are recommended.