Sperm cryopreservation is very important to genetic material quality dissemination. However, the spermatozoa can be damaged during this process. The objective of this study was to investigate the functionality of spermatozoa plasmatic membrane of ovine after cryopreservation. The work was conducted at Laboratory of Reproduction Biotechnology, Veterinary Medicine course, Federal University of Piaui. Fifteen animals were used (5 Dorper, 5 Santa Inês, and 5 animals of undefined breed - SRD). The animals were between 2 and 3 years old, and only one ejaculate was used per animal. Semen was collected via artificial vagina and frozen in cryopreservation machine (TK 3000^®, OV curve 2), with Tris-yolk base extend and glycerol. The semen was evaluated before and after cryopreservation. To investigate the functionality of spermatozoa plasmatic membrane hypo-osmotic test (HOST) was used. After thawing, 20 μL of semen was incubated in 2 mL of hypoosmotic solution (125 mOsmol L^-1) and 20 μL of semen was incubated in a 4% formalide citrate solution at 37°C for 1 h. After incubations, both samples were placed on microscopic slides, and 200 cells were visually evaluated per slide. Data were analyzed by Tukey-Kramer test (parametric data) and Dunn test (no parametric data) by Grafpad Instat^® software. The motility averages after thawing were 36 ± 15.2% (Dorper), 36 ± 19.5% (Santa Inês), and 21 ± 14.3% (SRD). If the plasma membrane over the principal piece is intact, the membrane will swell, causing the tail to coil, whereas spermatozoa with damaged principal piece membranes will not swell. The HOST averages were 24.2 ± 11.8% (Dorper), 21% ± 6.4 (Santa Inês), and 10.4 ± 1.6% (SRD). Motility and HOST showed positive correlation (P < 0.001). The SRD animals showed higher correlation numbers than other groups (P < 0.009). The HOST is a cheap, easy, and efficient technique to estimate the functionality of sperm plasmatic membrane. We suggest that other studies could be done with more animals and more samples.