The eutrophication of freshwater ecosystems allows the proliferation of cyanobacteria that can produce secondary metabolites such as microcystins. The main aim of this study was to explore the occurrence and concentration of microcystin and the mcyA gene in water bodies located in agricultural, urban, and recreational areas in the karst aquifer of the Yucatan peninsula of Mexico (YPM) and to analyze the water quality variables and chlorophyll-a (Chl-a) associated with their presence. Water samples were collected from 14 sites, and microcystin concentrations were quantified using antibody-based ELISA test. Total DNA was isolated from filters and used for PCR amplification of a fragment of the mcyA gene. Amplicons were cloned and sequenced to identify toxin-producing cyanobacteria present in water. Results showed that water bodies had different trophic status based on Carlson’s trophic state index. Dissolved inorganic nitrogen (DIN: NH₄⁺ + NO₃⁻ + NO₂⁻) and P-PO₄³⁻ concentrations were within a range of 0.077–18.305 mg DIN/L and 0.025–2.5 mg P-PO₄³⁻/L, respectively, per sampled site. All sampled sites presented microcystin concentrations within a range of ≥0.14 µg/L to ≥5.0 µg/L, from which 21.4% (3/14) exceeded the limit established in water quality standards for water consumption (1 µg/L). The mcyA gene fragment was detected in 28.5% (4/14) of the sites. A total of 23 sequences were obtained from which 87% (20/23) shared >95% nucleotide identity (nt) with the genus Microcystis and 13% (3/23) shared >87% nt identity with uncultured cyanobacteria. No correlation with the presence of the mcyA gene and microcystins was found; however, a positive correlation was detected between microcystin concentrations with pH and Chl-a.