The carbapenemase OXA-48 was identified for the first time in 2001 and is now one of the greatest concerns in terms of antibiotic resistance. While many studies report clinical OXA-48-like producers, few reports refer blaOXA-48-like genes in environmental bacteria. The main goal of this study was to evaluate the diversity of blaOXA-48-like genes in aquatic systems, using culture-independent approaches. For that, environmental DNA was obtained from riverine and estuarine water and used to construct clone libraries of blaOXA-48-like gene polymerase chain reaction amplicons. blaOXA-48-like libraries from river and estuarine water DNA comprised 75 and 70 clones, respectively. Sequence analysis showed that environmental blaOXA-48-like genes show a broader diversity than that so far observed in clinical settings. In total, 50 new OXA-48 variants were identified as well as sequences identical to previously reported OXA-48, OXA-181, OXA-199, OXA-204 and OXA-162. Though we have no evidence that these genes were carried by bacteria that are members of the natural heterotrophic flora or bacteria that have entered this particular water environment through anthropogenic sources, these results reinforce the role of aquatic systems as antibiotic resistance reservoirs. The variants of blaOXA-48 here described should be taken into account when designing molecular strategies for detecting this gene.