Abstract - The comparison of one and two-step dilution techniques of frozen semen was scrutinized based on post- thaw sperm quality and the fertility rate of cows after insemination in natural heat. The semen characteristics of two different pure breeds and two different crossbreed bulls, namely Harison (Friesian 100%), George (Friesian X), Bahadur (Sahiwal 100%) and Lalchand (Sahiwal X) were compared. Semen was diluted in a Tris-based extender with glycerol containing 20% egg yolk to obtain 20 ? 106 spermatozoa per milliliter. In one step dilution, TRIS-egg yolk extender was added at 37?C, and in two steps dilution, TRIS-egg yolk extender with glycerol was added at 5?C. After two hours of equilibration with glycerol, all semen samples were frozen by box method placing the straws horizontally above the liquid N2 surface in a Styrofoam box. Sperm motility was assessed by using a CASA system before and after thawing. Results indicated that semen collected from individual bulls varies in quality and kinetic velocity but these variations were insignificant. In addition, the parameters of semen quality have been reduced after freezing and thawing but most of these variations were insignificant between dilution techniques. On the other hand, no significant variation was observed in pregnancy rates considering the bulls and dilution methods. So, the one-step dilution method could be a method of choice in the processing of semen during cryopreservation because of its easy operating technique than the two-step dilution method.