OBJECTIVE: This research aims to investigate the epidemiology and genetic changes of Shigella species in the cities of Qazvin and Tehran from 2003 to 2023, with a focus on assessing antibiotic resistance among the isolates. This study is based on the analysis of 80 Shigella isolates, which were obtained from patients' feces. RESULTS: Using pulsed-field gel electrophoresis (PFGE) as the primary typing method, the isolates were classified into 13 distinct clusters, revealing a dominant pattern that has persisted for over twenty years. This persistence may be attributed to factors such as poor hygiene, personal contact, immigration, and antibiotic resistance. Genetic analysis indicated clonal expansion among the isolates over the two-decade period. Notably, the highest resistance was observed against amoxicillin, with several isolates exhibiting multidrug resistance.