ABSTRACTMultidrug-resistantVibrio choleraeO1 strains have long been observed in Africa, and strains exhibiting new resistance phenotypes have emerged during recent epidemics in Kenya. This study aimed to determine the epidemiological aspects, drug resistance patterns, and genetic elements ofV. choleraeO1 strains isolated from two cholera epidemics in Kenya between 2007 and 2010 and between 2015 and 2016. A total of 228V. choleraeO1 strains, including 226 clinical strains isolated from 13 counties in Kenya during the 2007–2010 and 2015–2016 cholera epidemics and two environmental isolates (from shallow well water and spring water isolates) isolated from Pokot and Kwale Counties, respectively, in 2010 were subjected to biotyping, serotyping, and antimicrobial susceptibility testing, including the detection of antibiotic resistance genes and mobile genetic elements. AllV. choleraeisolates were identified as El Tor biotypes and susceptible to ceftriaxone, gentamicin, and ciprofloxacin. The majority of isolates were resistant to trimethoprim-sulfamethoxazole (94.6%), streptomycin (92.8%), and nalidixic acid (64.5%), while lower resistance was observed against ampicillin (3.6%), amoxicillin (4.2%), chloramphenicol (3.0%), and doxycycline (1.8%). Concurrently, the integrating conjugative (SXT) element was found in 95.5% of theV. choleraeisolates; conversely, class 1, 2, and 3 integrons were absent. Additionally, 64.5% of the isolates exhibited multidrug resistance patterns. Antibiotic-resistant gene clusters suggest that environmental bacteria may act as cassette reservoirs that favor resistant pathogens. On the other hand, the 2015–2016 epidemic strains were found susceptible to most antibiotics except nalidixic acid. This revealed the replacement of multidrug-resistant strains exhibiting new resistance phenotypes that emerged after Kenya's 2007–2010 epidemic.IMPORTANCEKenya is a country where cholera is endemic; it has experienced three substantial epidemics over the past few decades, but there are limited data on the drug resistance patterns ofV. choleraeat the national level. To the best of our knowledge, this is the first study to investigate the antimicrobial susceptibility profiles ofV. choleraeO1 strains isolated from two consecutive epidemics and to examine their associated antimicrobial genetic determinants. Our study results revealed two distinct antibiotic resistance trends in two separate epidemics, particularly trends for multidrug-associated mobile genetic elements and chromosomal mutation-oriented resistant strains from the 2007–2010 epidemic. In contrast, only nalidixic acid-associated chromosomal mutated strains were isolated from the 2015–2016 epidemic. This study also found similar patterns of antibiotic resistance in environmental and clinical strains. Continuous monitoring is needed to control emerging multidrug-resistant isolates in the future.