Different nematodes affect canines, however Acanthocheilonema reconditum was considered mostly a non-pathogenic parasite. Climate change, animal migration, and other factors transformed the dynamics of vector-borne diseases, including filariasis. Since 2016, a sudden increase in the number of dogs with microfilaremia was reported by different veterinary centers in Cali, southwest Colombia. The objective of this study was to molecularly identify the etiologic agent of this filariasis outbreak detected in this city, using PCR–RFLP and evaluating dogs’ clinical signs. From 2018–2019, canine filariasis cases were prospectively evaluated after a microscopic test, recruiting 82 cases and 43 healthy controls from 2971 samples. Acanthocheilonema reconditum (Nematoda, Onchocercidae) was identified in 61.3% of the cases (49/82) by PCR–RFLP. Sanger sequencing of the 5.8S ribosomal RNA gene and internal transcribed spacer-2 fragment was additionally performed on seven cases, confirming A. reconditum in all of them. The filariasis cases are statistically associated with male dogs who have clinical signs of anemia, low levels of hemoglobin and hematocrit (p < 0.0001), and high levels of plasma proteins (p < 0.001). This emerging canine disease constitutes an important public health concern among veterinarians and active surveillance is advised to explore its zoonotic potential.