BACKGROUNDS: Sperm sexing as one of the important ways for pre-selection of offspring along with artificial insemination, has the potential to considerably improve animal breeding and the efficiency of dairy and meat production. The discrepancy between X- and Y- chromosome bearing sperm is the basis of this procedure. OBJECTIVES: The aim of this study was to evaluate a discontinuous sperm isolation medium Allgrad gradient in separation of buffalo spermatozoa according to sex chromosomes by fluorescence in-situ hybridation (FISH). METHODS: A four-layer discontinuous Allgrad gradient, 65%-95% with 10% differences between each subsequent layer was prepared in gradient separation for post thaw X- and Y-bearing spermatozoa. Following centrifugation, sperm from the top and bottom fraction were aspirated and fixed on a slide. The FISH procedure was performed by using X and Y bovine specific DNA probes. At least 400 spermatozoa were scored for each sample. The proportions of X- and Y-bearing spermatozoa were determined by the presence of red or green fluorescent signals. RESULTS: There was no significant difference in the percentage of spermatozoa with the specific signals of X and Y chromosomes in the control groups. After separation, in treatment groups, the percentage of X-bearing spermatozoa in the bottom layer exceeded that of the one in the top layer (78.3 vs. 21.7) (p<0.05). Adversely, in all treatment groups the significant difference between the frequencies of Y-bearing spermatozoa in the top layers was evident (75.5vs.24.5)(p<0.05). CONCLUSIONS: This study showed that using four-step discontinuous gradient by Allgrad isolation medium was a reliable method for the separation of X- and Y-bearing frozen-thawed buffalo spermatozoa and can be more expedient for IVF in Buffalo.