Background and Aim: Canine vector-borne diseases represent an important issue for the welfare and health of animals, but also have great zoonotic potential. These diseases are caused by bacteria, nematodes such as filariae, and other parasites such as Leishmania spp. Given the difficulty in differentiating common microfilariae in dogs by microscopy and serological methods, molecular techniques such as polymerase chain reaction (PCR) and sequencing should be valuable for reaching a reliable diagnosis. This study aimed to use microscopy and PCR to identify the microfilarial species in dogs from Valle del Cauca, Colombia, and a possible association with Leishmania infantum parasites. Materials and Methods: This study was conducted on 270 dogs from Pradera and Florida municipalities. Microfilariae were detected in dogs by optical microscopy and amplification with 5.8S-ITS2-28S. Species identification was achieved through the amplification of the gene cytochrome oxidase I (COX1). Results: Microscopic detection of microfilariae was possible in 4.81% (13/270) of the dogs. In addition, by PCR of COX1 and Sanger sequencing of ITS2, Acanthocheilonema reconditum was identified as the circulating microfilarial species in 12 dogs, coinfecting with the species L. infantum (Leishmania donovani complex). Conclusion: To the best of our knowledge, this is the first report on A. reconditum and L. infantum mixed infection in dogs in Colombia, particularly in the Valle del Cauca. [ABSTRACT FROM AUTHOR]