Fewer studies investigate the effects of underlying genetic factors related to semen characteristics, significantly affecting sheep farm profitability. This study aimed to identify single nucleotide polymorphisms (SNP) and genomic regions associated with fresh and frozen-thawed semen traits in rams with low (Hasak) and high (Hasmer) cryotolerance. Semen collected from 11 (5 Hasak with low and 6 Hasmer with high cryotolerance) rams cryopreserved in 0.25 ml straws in the breeding season. Quality characteristics were determined in fresh, equilibrated, and frozen-thawed semen. A Genome-Wide Association Study (GWAS) was conducted to unveil the genetic structure that might be attributed to cryotolerance in low and high cryotoleranced rams. Fresh (regarding total and progressive motility) and equilibrated semen quality were similar in Hasak and Hasmer rams (p > 0.6). However, the freeze-thawing process had a more pronounced negative effect on ram semen traits in Hasak than in Hasmer (p < 0.05). GWAS revealed 27 SNPs correlated with post-thaw semen parameters. Moreover, network analyses revealed pathways related to sperm ion channels and their activities, providing insights into the intricate molecular mechanisms underlying sperm physiology and emphasizing their role in potentially impacting sperm cryotolerance. The functional significance of detected SNPs and the associated pathways require further exploration.