Genome-wide association studies (GWAS) are useful for nominating candidate genes, but typically are unable to establish disease causality or differentiate between the effects of variants in linkage disequilibrium (LD). Additionally, some GWAS loci might contain multiple causative variants or genes that contribute to the overall disease susceptibility at a single locus. However, the majority of current GWAS lack the statistical power to test whether multiple causative genes underlie the same locus, prompting us to adopt an alternative approach to testing multiple GWAS genes empirically. We used gene targeting in a disease-susceptible rat model of genetic hypertension to test all six genes at the Agtrap-Plod1 locus (Agtrap, Mthfr, Clcn6, Nppa, Nppb, and Plod1) for blood pressure (BP) and renal phenotypes. This revealed that the majority of genes at this locus (five out of six) can impact hypertension by modifying BP and renal phenotypes. Mutations of Nppa, Plod1, and Mthfr increased disease susceptibility, whereas Agtrap and Clcn6 mutations decreased hypertension risk. Reanalysis of the human AGTRAP-PLOD1 locus also implied that disease-associated haplotype blocks with polygenic effects were not only possible, but rather were highly plausible. Combined, these data demonstrate for the first time that multiple modifiers of hypertension can cosegregate at a single GWAS locus.