Samples were collected from 128 symptomatic humans, 83 dogs, 49 mice, and 20 rats ( Rattus rattus : 16; Rattus norvegicus : 4) in neighborhoods where human leptospirosis have been reported within the principal sea-port city of Colombia. Seroprevalences were assessed against 19 pathogenic, 1 intermediate pathogenic, and 1 saprophytic Leptospira serogroups. Pathogenic Leptospira were confirmed using conventional Leptospira -specific polymerase chain-reaction and pulsed-field gel electrophoresis analysis was used for serovar identification. Seroprevalences of 20.4%, 12.5%, 25.0%, 22.9%, and 12.4% were obtained against one to seven different serogroups in mice, R. rattus , R. norvegicus , dogs, and humans, respectively. The DNA was confirmed to be from pathogenic Leptospira by detecting the lipL32 gene in 12.5%, 3.7%, and 0.03% of the R. rattus , dog, and human samples, respectively. The first genetically typed Colombian isolate was obtained from a rat and identified as Leptospira interrogans serovar Icterohaemorrhagiae/Copenhageni.