Klebsiella spp. is recognized as an emerging threat to public health worldwide mainly due to its increasing prevalence in healthcare-associated infections caused by multidrug-resistant strains producing extended-spectrum β-lactamases (ESBL). However, comparable data on its occurrence in surface waters and hospital wastewater are scarce. Therefore, we aimed to investigate the prevalence of ESBL-producing Klebsiella spp., in surface waters and hospital wastewater, and characterize the isolates phenotypically and genotypically. Seventy-seven samples were collected from different surface waters in the North of Portugal, of which 35 (45,5%) were positive for Klebsiella spp. However, 44 samples were collected from hospital wastewater in northern Portugal, of which 40 (90.9%) were positive for Klebsiella spp. The antimicrobial susceptibility was performed using the Kirby-Bauer disk diffusion method against 11 antibiotics. Screening for ESBL phenotypic production was performed by double-disk synergy. ESBL production was detected in 11 (31,4%) of 35 Klebsiella spp. isolated in surface waters, and in 20 (50%) of 40 Klebsiella spp., in hospital wastewater. The presence of different carbapenem genes (e.g., blaKPC) was analyzed by PCR in Klebsiella spp. isolates, as well as the presence of other resistance genes (e.g., blaCTX and blaSHV). The one with the highest prevalence, in the surface waters, was blaCTX (22,6%) however, in hospital wastewater, was blaSHV (52,5%). Nevertheless, in carbapenem genes, amplification was not verified, in surface waters, but in hospital wastewater, was verified in the blaKPC (10%). Monitoring the evolution of the ESBL situation and applying a One Health approach is essential to keep this problem under control.