Male infertility is an emerging problem in both humans and animals, and the knowledge of its causes is the first step to identifying new diagnostic and therapeutic strategies. In humans, alteration of sperm DNA methylation have been related to poor quality semen, impaired seminal parameters, azoospermia and reduced fertility. Although semen analysis is routinely used to evaluate the male reproductive potential in the canine species, no authors have attempted to relate semen characteristics to the sperm global DNA methylation (SGDM). The aim of this study was to evaluate the SGDM level in healthy dogs and to correlate it with semen parameters that are currently used in dog semen analyses. Conventional and unconventional (sperm DNA fragmentation and SGDM) seminal parameters of thirty dogs from different breeds were evaluated. A positive correlation was found between SGDM and sperm concentration (r = 0.41; p < 0.05), and total sperm count (r = 0.61; p < 0.001); SGDM was significantly lower in oligozoospermic vs non-oligozoospermic dogs (4.3% vs. 8.7%; p < 0.005). Our findings suggest that SGDM levels are related to conventional seminal parameters, and could be used as a marker of testis function and spermatogenesis in dogs.