The aim of this study was to analyse and identify specific buffalo seminal plasma proteins (SPPs) responsible for sperm cryotolerance during low temperature storage. Computer Assisted Sperm Analysis (CASA) of the motility and viability of buffalo spermatozoa was performed before freezing and after thawing. Two sample groups were formed – ejaculates with high cryotol- erance (group A) and low cryotolerance (group B). CASA demonstrated that the initial progres- sive motility after thawing of the spermatozoa in group A is significantly higher than in group B (p<0.001). Group B showed a significant increase in the percentage of static and non-progressive spermatozoa at 240 min, when compared to group A (p<0.05). SPPs, proteins in the cryoprotec- tive medium (PM) and proteins in the mixture of PM and SP were separated by High Perfor- mance Liquid Chromatography (HPLC). Comparative analysis of the chromatographic profiles was performed to identify specific proteins related to sperm cryotolerance. SPPs profiles showed 5 distinct protein peaks in both groups, ranging from 500 kDa to 50 Da. Chromatograms of group A and group B showed quantitative and qualitative differences in protein content. Chromato- grams of proteins in PM showed 11 well-expressed peaks. HPLC analysis of the mixtures of SPPs from the two groups and PM visualized the formation of a new bio-complex structure expressed by a protein peak specific for group A (7.674 min, AU 1.50). This protein peak can be referred as a phenotypic trait for buffalo ejaculates with high sperm cryotolerance.