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In the department of Boyacá, Colombia, agriculture stands as one of the primary economic activities. However, the escalating utilization of pesticides within this sector has sparked concern regarding its potential correlation with elevated risks of genotoxicity, chromosomal alterations, and carcinogenesis. Furthermore, pesticides have been associated with a broad spectrum of genetic polymorphisms that impact pivotal genes involved in pesticide metabolism and DNA repair, among other processes. Nonetheless, our understanding of the genotoxic effects of pesticides on the chromosomes (as biomarkers of effect) in exposed farmers and the impact of genetic polymorphisms (as susceptibility biomarkers) on the increased risk of chromosomal damage is still limited. The aim of our study was to evaluate chromosomal alterations, chromosomal instability, and clonal heterogeneity, as well as the presence of polymorphic variants in the GSTP1 and XRCC1 genes, in peripheral blood samples of farmers occupationally exposed to pesticides in Aquitania, Colombia, and in an unexposed control group. Our results showed statistically significant differences in the frequency of numerical chromosomal alterations, chromosomal instability, and clonal heterogeneity levels between the exposed and unexposed groups. In addition, we also found a higher frequency of chromosomal instability and clonal heterogeneity in exposed individuals carrying the heterozygous GSTP1 AG and XRCC1 (exon 10) GA genotypes. The evaluation of chromosomal alterations and chromosomal instability resulting from pesticide exposure, combined with the identification of polymorphic variants in the GSTP1 and XRCC1 genes, and further research involving a larger group of individuals exposed to pesticides could enable the identification of effect and susceptibility biomarkers. Such markers could prove valuable for monitoring individuals occupationally exposed to pesticides.