Since the beginning of the SARS-CoV-2 outbreak, different animal species have been implicated as possible intermediate hosts that could facilitate the transmission of the virus between species. This is the reason why the detection of these hosts has intensified, revealing a series of reports involving wild, zoo, farm, and pet animals. The goal of the present study was to determine the seroprevalence of anti-SARS-CoV-2 immunoglobulins (Ig) class G (IgG) in domestic dogs and cats and its epidemiological association with the frequency of COVID-19 patients in the city of Villavicencio, Colombia. To that end, 300 domestic canines and 135 domestic felines were randomly selected in a two-stage distribution by clusters according to the presence of active COVID-19 cases (positive RT-qPCR for SARS-CoV-2) within the human population distributed within the eight communes of the urban centre of the city of Villavicencio, Colombia. The indirect ELISA technique was applied in order to determine the presence of anti-SARS-CoV-2 IgG antibodies in sera samples. Kernel density estimation was used to compare the prevalence of COVID-19 in humans by commune with the seropositivity of domestic dogs and cats found in this study. The overall seroprevalence of anti-SARS-CoV-2 IgG was 4.60% (95% CI = 3-7). Specifically in canines, the results indicated 3.67% (95% CI = 2.1-6.4), and in felines 6.67% (95% CI = 3.6-12.18). Kernel density estimation indicated that seropositive cases were concentrated in the west region of the city (communities 2 and 4). Likewise, there was a positive association between SARS-CoV-2 seropositivity in pet animals and their habitat in Commune 1 (adjusted OR = 5.84; 95% CI = 1.1-30.88). Spearman's correlation coefficients were weakly positive ( p = 0.32) between the ratio of COVID-19 RT-qPCR for positive cases in humans in November 2020 in comparison to domestic dogs and cats from the eight communes of Villavicencio sampled in the present study, which provided the first positive results of anti-SARS-CoV-2 ELISA serological tests in domestic dogs and cats in Colombia with information about the dynamics of the virus transmission in Latin America and the world during the COVID-19 pandemic.