Wildlife-vehicle collisions are acknowledged as the leading source of vertebrate mortality worldwide. Here, we examined spatio-temporal patterns of mammal road mortality in three routes on a fragmented landscape in the Middle Magdalena Valley, Santander department, Colombia. We identified the species affected by road mortality, estimated the roadkill rates for each road segment, and identified roadkill hotspots related to multi-scale structural connectivity. During an eigth-month period, we recorded a total of 152 mammal roadkills of 12 native species. The roads with the highest roadkill rates were Departmental Route 01 (DR01) and National Route 66 (NR66), but roadkill aggregations corresponding to potential roadkill hotspots were only identified on the route DR01. These hotspots intersect with sections of the landscape with lower connectivity, suggesting that mammal species are forced to leave suitable habitats in response to habitat fragmentation, increasing the risk of wildlife-vehicle collisions. Our results contribute to understanding spatio-temporal factors potentially promoting mammal roadkill events and offer a unique opportunity to designing effective mitigation measures that will work properly.