Abstract: The objective of the present study was to investigate the possibility of preserving mithun (Bos frontalis) spermatozoa at refrigeration temperature using tris–egg yolk diluent. Semen samples were collected from four adult mithun bulls through rectal massage method. Good quality semen samples (n =30) were preserved at 4°C using tris–egg yolk diluent for 72h. Progressive motility, live spermatozoa count and morphological abnormalities were evaluated every 12h until 72h of preservation. The colour, consistency and mass activity of fresh semen samples were found to be creamy white, medium and 3+ to 4+ (5+ scale), respectively. The average (mean±S.E.) volume (ml), pH and spermatozoa concentration (106 ml−1) of fresh semen samples were found to be 0.6±0.01, 6.8±0.03 and 425±48, respectively. Progressive motility and live spermatozoa count were found to be less than 30% (P <0.01) after 48h of storage. Head (P <0.05), midpiece (P <0.05), tail (P <0.01) and total (P <0.01) abnormalities were found to be increased significantly over the time of storage. It was observed that progressive motility and live spermatozoa count remained above 30% and 40%, respectively, until 36h of storage. Simultaneously the percentage of morphologically abnormal spermatozoa was found to be significantly low until 36h of storage. The results indicate that it is possible to preserve mithun spermatozoa at refrigeration temperature in tris–egg yolk diluent, which can be further used for artificial insemination within 36h of storage. [Copyright &y& Elsevier]