The increase in Klebsiella spp. multidrug-resistant strains represent a serious threat to public health. Thus, currently human, and veterinary medicine is threatened worldwide by a growing resistance to antibiotics, particularly present in opportunistic Enterobacterial pathogens (e.g., Klebsiella spp.). However, comprehensive, and comparable data on its occurrence in surface waters are scarce, as well as phenotypic and genotypic characteristics for several countries including Portugal. Given this scenario, we aimed to investigate the prevalence of Klebsiella spp. in surface waters, as well as characterize the isolates phenotypically and genotypically. Sixty-five samples were collected from different surface waters in the North of Portugal, of which 31 (47,7%) were positive for Klebsiella spp. The antimicrobial susceptibility was performed by the Kirby-Bauer disc diffusion method against 11 antibiotics. High rates of antibiotic resistance were observed among these isolates for cefotaxime (41,9%), on the other hand, low rates of antibiotic resistance were observed among these isolates for meropenem (3,2%). The presence of different carbapenem genes (blaOXA, blaIMP, and blaKPC) was analyzed by PCR/sequencing in Klebsiella spp. isolates, as well as the presence of other resistance genes (e.g. blaCTX). The one with the highest prevalence was blaCTX (22,6%). However, in the carbapenem genes, amplification was not verified. Our study provides information on the prevalence of Klebsiella spp., which is of clinical importance in surface water in Portugal. These findings indicate their possible dissemination in the environment and the potential risk of colonization and/or infection of humans and livestock associated with exposure to contaminated water sources.