We compared the resistomes within polluted and unpolluted rivers, focusing on extended-spectrum beta-lactamase (ESBL) genes, in particular bla(CTX-M). Twelve rivers from a Portuguese hydrographic basin were sampled. Physicochemical and microbiological parameters of water quality were determined, and the results showed that 9 rivers were classified as unpolluted (UP) and that 3 were classified as polluted (P). Of the 225 cefotaxime-resistant strains isolated, 39 were identified as ESBL-producing strains, with 18 carrying a bla(CTX-M) gene (15 from P and 3 from UP rivers). Analysis of CTX-M nucleotide sequences showed that 17 isolates produced CTX-M from group 1 (CTX-M-1, -3, -15, and -32) and 1 CTX-M that belonged to group 9 (CTX-M-14). A genetic environment study revealed the presence of different genetic elements previously described for clinical strains. ISEcp1 was found in the upstream regions of all isolates examined. Culture-independent bla(CTX-M)-like libraries were comprised of 16 CTX-M gene variants, with 14 types in the P library and 4 types in UP library, varying from 68% to 99% similarity between them. Besides the much lower level of diversity among CTX-M-like genes from UP sites, the majority were similar to chromosomal ESBLs such as bla(RAHN-1). The results demonstrate that the occurrence and diversity of bla(CTX-M) genes are clearly different between polluted and unpolluted lotic ecosystems; these findings favor the hypothesis that natural environments are reservoirs of resistant bacteria and resistance genes, where anthropogenic-driven selective pressures may be contributing to the persistence and dissemination of genes usually relevant in clinical environments.