This study aimed to assess the suitability of egg yolk (EY) supplementation to a tris–citric acid–based extender on cryosurvival of guinea pig ( Cavia porcellus ) epididymal spermatozoa. Two synthetic-based extenders, tris–citric acid–glucose plus 20% EY (TCG-EY) and tris–citric acid–fructose (TCF) both with 5% glycerol, were compared. Thirty-two epididymides were recovered from 16 adult guinea pig males by gonadectomy, and then the sperm samples were retrieved by retrograde flushing using TCG-EY and TCF extenders for left or right epididymis, respectively. TCG-EY and TCF sperm samples were frozen in static liquid nitrogen vapors through a two-step cooling procedure. Before freezing, the percentage of progressive sperm motility and sperm with intact plasma and acrosome membranes from TCG-EY sperm samples were higher ( p < 0.05) than those diluted with TCF. Post-thaw sperm kinematic variables and membrane integrity were drastically reduced ( p < 0.001) compared with prefreezing samples, regardless of extender type. The post-thaw plasmatic and acrosome membrane integrity from TCG-EY sperm samples was higher ( p < 0.05) than those from TCF samples. Except for the length, the morphometric head dimensions of sperm diluted with TCG-EY or TCF did not vary ( p > 0.05) after the freezing–thawing process compared with the prefreezing samples. In conclusion, despite greater cell cryoinjury with both extenders, the EY supplementation exerted greater cell membrane protection before and after the freezing–thawing process. This research shows an in-depth analysis of guinea pig sperm cryopreservation; however, more studies are recommended.