Diarrhea in alpaca crias significantly impacts livestock health in high-altitude regions, with Escherichia coli as a common pathogen. This study analyzed 10 E. coli isolates from diarrheic and healthy alpacas using whole-genome sequencing to assess genetic diversity, virulence factors, and antibiotic resistance. Predominant sequence types (ST73, ST29), serotypes (O22:H1, O109:H11), and phylogroups (B2, B1, A) were identified. Virulence profiling revealed ExPEC-like and EPEC pathotypes, while resistance genes for β-lactams (blaEC-15), fosfomycin (glpT_E448K), and colistin (pmrB) were prevalent. These findings highlight the need for genomic surveillance and antimicrobial stewardship to manage E. coli infections in alpacas and reduce public health risks.