Objective: To evaluate frequency, distribution and resistance to s-lactamic antibiotics in isolations of Klebsiella pneumoniae (Kpn), Klebsiella oxytoca (Kox) y Escherichia coli (Eco), and characterize s-lactamases responsible for such resistance. Methods: 614 strains (107 Kpn, 493 Eco y 14 Kox) strains were selected, isolated from patients, between January and December 2013. Identification and phenotypic analysis was performed through MicroScan method automated systems. ESBL was confirmed through double-disk method with cefotaxime in front of cefotaxime plus clavulanate and ceftazidima in front of ceftazidima / clavulanate according to the CLSI. Molecular detection of CTX-M gens was detected by using specific primers. Results: 15,96% of total isolations (98/614), corresponded to ESBL. Eco was the most common specie in production of them, with 10.6% (65/614). ESBL production was observed in 14,3% (14/98) of intra-hospital isolations, 46,9% (46/98) was detected at the emergency clinic, and 60,2% (59/98) of positive ESBL cultures corresponded to urine samples; 65.3% (64/98) strains produced ESBL CTX-M; 51% (50/98) ESBL TEM and 51% (50/98) were ESBL SHV producers. Conclusions: The study showed a high frequency of ESBL CTX-M and ESBL TEM in Eco, Kox, and Kpn. The strains were isolated mainly from the emergency clinic, and in patients with urinary infection.