<p>Urbanization, characterized by increased temperatures and habitat fragmentation, poses significant threats to reptile populations. Temperature threatens reptile populations, reducing the available hours of activity; however, the deleterious effects of temperature on sperm integrity have been scarcely investigated. This study investigates the impact of urbanization on the sperm fertility of the mesquite lizard (<i>Sceloporus grammicus</i>) in the Mexican Megalopolis. The study focused on sites with varying degrees of urbanization, analyzing sperm cell viability, motility, and concentration, as well as DNA integrity and cytoplasmic droplets, during the reproductive season of lizards. Additionally, we measured the thermal quality of the environment as a means to assess temperature variation within the gradient. The results showed that sperm viability decreased with increased urbanization levels across all the epididymides. DNA integrity was also adversely affected in the caput and corpus regions, but not in the cauda. The study found that urban areas have an adverse impact on sperm viability, potentially compromising reproductive capacity. These findings underscore the need for additional research on the effects of anthropogenic factors on reptile reproduction and emphasize the importance of implementing conservation strategies to mitigate these impacts.</p>