Background: In recent years, the prevalence of antibiotic resistance has steadily increased and also the antibiotic-resistant strains producing extended-spectrum beta-lactamases (ESBLs) and AmpC beta-lactamases have emerged among the Enterobacteriaceae, predominantly in Escherichia coli and Klebsiella pneumoniae species. Objectives: This prospective study aimed at determining the production of ESBL or AmpC, phenotypically and also at the molecular level, in E. coli and K. pneumoniae isolates collected from various clinical specimens. Methods: In total, 78 K. pneumoniae and 92 E. coli isolates were collected from various clinical infectious sources available in different wards of the Imam Reza Hospital, Tabriz, Iran, from July 2017 to December 2018. All isolates were subjected to antimicrobial susceptibility testing. MAST 4-disc test and polymerase chain reaction (PCR) were applied for phenotypic and genotypic detection of ESBLs and plasmid-encoded AmpCs (pAmpCs) among isolates, respectively. Results: Overall, 78 K. pneumoniae and 92 E. coli isolates were evaluated, of which 46 K. pneumoniae (58.9%) and 51 E. coli (55.4%) isolates were resistant to cefotaxime/ceftazidime and included in the study. Among the K. pneumoniae and E. coli isolates resistant to cefotaxime/ceftazidime, 40 (86.9%) and 40 (78.4%) isolates were ESBL producers and 8 (17.3%) and 2 (3.9%) isolates were pAmpC producers, respectively. In addition, 40 E. coli (78.4%) isolates were positive for both CTX-M-14 and CTX-M-15 genes. Regarding K. pneumoniae isolates, 40 isolates (86.9%) were positive for CTX-M-15 gene and 18 isolates (39.1%) for CTX-M-14 gene. Among 51 ceftazidime/cefotaximeresistant E. coli isolates, 32 isolates (62.7%) were positive for DHA-1 gene and 33 isolates (64.7%) isolates for CMY-2 gene. Also, among 46 ceftazidime/cefotaxime-resistant K. pneumoniae isolates, 15 isolates (32.6%) had DHA-1 gene and 27 isolates (58.7%) had CMY-2 gene in the genome. Conclusions: The high prevalence of ESBL and AmpC production among E. coil and K. pneumoniae isolates was a serious concern in the studied region. Therefore, a simple and rapid PCR-based technique is essential to detect and distinguish various pAmpC and ESBL genes to discriminate other resistance determinants.