Urban wastewater treatment plants (UWTPs) are considered hot spots for the accumulation and transfer of antibiotic resistant bacteria and their genes. We investigated the prevalence, diversity, and genomic localization of β-lactamase resistance genes in wastewater from different parts of an UWTP, such as raw wastewater, wastewater from biological reactor, and treated wastewater, collected from the UWTP of Warsaw, Poland. In this study, we focused on gram-negative rods, mainly Enterobacteriaceae, and used Multiplex PCR to identify various β-lactamase resistance genes (bla). Susceptibility to a number of antibiotics and genomic localization of β-lactamase resistance genes were then determined using disc-diffusion and Southern hybridization methods, respectively. No differences in the frequency of different types of bla genes between the sampling points were discerned. Three new variants of β-lactamase genes (bla CMY-157 , bla MOX-13 , and bla FOX-15 ) were identified. Four bla genes (bla TEM-12 , bla TEM-30 , bla TEM-47/68 , bla ACT ) that had never been found in UWTPs were identified in this study. Nine of the identified bla genes had been found in the same environment previously. The bla FOX-15 variant on a Kluyvera sp. plasmid and bla GES type on Raoultella spp. plasmids were observed for the first time in these genera. There was a decrease in the number of multidrug resistant strains in the effluent compared to the influent. Finally, a significantly higher number of cefotaxime and carbapenem unsusceptible strains were detected in the influent than in the effluent. The results strongly support the hypothesis of UWTPs as a hot spot for antibiotic resistant genes (ARGs) and antibiotic resistant bacteria (ARB) accumulation and indicate that β-lactamase genes are widely disseminated among gram-negative rods isolated from this environment.