Central Mexico has suffered accelerated rates of habitat fragmentation caused by the growth of agriculture, industry, and rural and urban settlements accompanied by a high density of roads. According to our hypothesis, the main highways in this region (Mex-057, Mex-047D, and Mex-045) have acted as barriers to dispersal and gene flow, resulting in a decrease of functional connectivity in the coyote (Canis latrans). In the period 2016–2020, we collected 537 scat samples from 13 sites in the state of Guanajuato and 33 sites in the state of Querétaro. Of these, 282 samples were from coyotes, which were identified through the sequencing of the cytochrome B gene. Based on 13 nuclear microsatellite loci and two sex-determining genes for canids (DBX6 and DBY7), we identified 66 distinct genotypes, 19 of which were females and 47 of which were males. The Bayesian analyses and spatial principal component analysis (sPCA) identified two genetic clusters (Gst = 0.072, p = 0.000), in which geographical relationship was not evident. We found moderate levels of genetic diversity (Ho = 0.633 ± 0.053) and few transient individuals (n = 10) were detected. Landscape resistance optimization analysis in ResistanceGA did not reveal the influence of major highways and land cover on genetic differentiation between individuals. According to our findings, major highways in Guanajuato and Querétaro do not restrict gene flow nor influence genetic structure; however, we cannot discard that the barrier effect is not yet detectable in the coyote population. © 2023, The Author(s), under exclusive licence to Mammal Research Institute Polish Academy of Sciences.
