This study evaluated associations between the presence of protein bands in ram seminal plasma and the quality of sperm frozen with distinct extenders. Ejaculates were frozen in a tris-egg yolk based extender, including either 5% glycerol or 100 mM trehalose. Seminal plasma samples were submitted to unidimensional electrophoresis. Pre-freezing and post-thawing sperm quality was similar between extenders (P > 0.05). A total of 26 bands were identified in ram seminal plasma. Pre-freezing sperm motility was increased when the 15, 19 and 80 kDa bands were present in seminal plasma (P < 0.05). The presence of an 11 kDa band in seminal plasma was associated with reduced pre-freezing membrane integrity (P < 0.05). After thawing, both sperm motility and membrane integrity were reduced when a 24 kDa band was present in seminal plasma (P < 0.05). Post-thawing acrosome integrity was greater in the presence of a 31 kDa band in seminal plasma (P < 0.05). Regardless of the cryoprotectant included in the freezing extender, these six bands may be potential markers for ram sperm tolerance to freezing