Objective The objective of this study is to determine the prevalence of rectal carriage of plasmid- and chromosome-encoded AmpC β-lactamase-producing Escherichia coli and Klebsiella spp. in patients in a Dutch teaching hospital between 2013 and 2016. Methods Between 2013 and 2016, hospital-wide yearly prevalence surveys were performed to determine the prevalence of AmpC β-lactamase-producing E. coli and Klebsiella spp. rectal carriage. Rectal swabs were taken and cultured using an enrichment broth and selective agar plates. All E. coli and Klebsiella spp. isolates were screened for production of AmpC β-lactamase using phenotypic confirmation tests and for the presence of plasmid-encoded AmpC (pAmpC) genes. E. coli isolates were screened for chromosome-encoded AmpC (cAmpC) promoter/attenuator alterations. Results Fifty (2.4%) of 2,126 evaluable patients were identified as rectal carrier of AmpC β-lactamase- producing E. coli. No carriage of AmpC β-lactamase producing Klebsiella spp. was found. Nineteen (0.9%) patients harboured isolates with pAmpC genes and 30 (1,4%) patients harboured isolates with cAmpC promoter/attenuator alterations associated with AmpC β-lactamase overproduction. For one isolate, no pAmpC genes or cAmpC promotor/ attenuator alterations could be identified. During the study period, a statistically significant decline in the prevalence of rectal carriage with E. coli with cAmpC promotor/attenuator alterations was found (p = 0.012). The prevalence of pAmpC remained stable over the years. Conclusions The prevalence of rectal carriage of AmpC-producing E. coli and Klebsiella spp. in patients in Dutch hospitals is low and a declining trend was observed for E. coli with cAmpC promotor/ attenuator alterations.