<p>In Guatemala, there is a significant gap in our understanding of the prevalence of antibiotic resistance across different reservoirs likely to affect human exposures. We conducted a One Health study of produce, poultry, water, and humans within peri-urban and urban Guatemalan markets to characterize the prevalence of third-generation cephalosporin-resistant <i>Escherichia coli</i> (3GCR-EC). We screened a total of 132 samples for 3GCR-EC using selective media. Samples were collected across three markets in Guatemala and included produce (<i>n</i> = 32), public tap water (<i>n</i> = 30), commercial and non-commercial poultry (<i>n</i> = 40), and human fecal matter (<i>n</i> = 30). We measured phenotypic and genotypic resistance using culture-based methods and whole-genome sequencing. Among the 132 samples, 87.1% (<i>n</i> = 115) were positive for <i>E. coli</i>, and 67.4% (<i>n</i> = 43) were positive for 3GCR-EC. After removing duplicates, a total of 40 3GCR-EC isolates were sequenced from commercial chicken carcasses (<i>n</i> = 8), non-commercial chicken carcasses (<i>n</i> = 16), water (<i>n</i> = 4), and human feces (<i>n</i> = 12). Out of 11 unique extended-spectrum beta-lactamase genes, the most prevalent gene was <i>bla</i>_CTX-M-55. There was high clonal diversity, with 32 distinct sequence types (ST) detected. Clinically important pandemic lineages of <i>E. coli</i> were identified: ST10, ST648, and ST410. This study identified a high prevalence of antibiotic resistance across diverse samples within public Guatemalan markets. The frequent detection of 3GCR-EC, their resistance genes, and epidemiologically important lineages of <i>E. coli</i> identified from humans, produce, water, and poultry samples highlight the importance of implementing more extensive surveillance using a One Health approach to develop coordinated strategies to mitigate the spread of antibiotic resistance.</p>