BACKGROUND Ruminants serve as one of the most important reservoirs for pathogenic Escherichia coli. Infection with E. coli, a foodborne enteropathogen, can lead to asymptomatic infections that can cause life-threatening complications in humans. Therefore, from a clinical and human health perspective, it is important to know which virulence genes, phylogenetic groups, serogroups, and antibiotic resistance patterns are present in E. coli strains in yaks with diarrheic infections. METHODS Two-hundred and ninety-two rectal swabs were collected from diarrheic yaks in Qinghai Plateau, China. The antimicrobial sensitivity of each resulting isolate was evaluated according to the disk diffusion method, and different PCR assays were performed for the detection of virulence genes and different phylogroups. Additionally, strains were allocated to different serogroups based on the presence of O antigen via the slide agglutination method. RESULTS Among the E. coli isolates tested, most of the isolates were multidrug resistant (97%) and harbored at least one virulence gene (100%). We observed ten virulence genes (sfa, eaeA, cnf1, etrA, papC, hlyA, aer, faeG, rfc, and sepA), of which sfa was the most commonly found (96.9%). Significant positive associations between some resistance phenotypes and virulence genes were observed (P < 0.05, OR > 1). The majority of the E. coli isolates belonged to phylogroup A (79.5%), and the others belonged to phylogroups B1 (7.5%), D (4.1%), B2 (5.8%), and F (0.7%). Among all the E. coli strains tested, serogroups O91 and O145 were the most prevalent, accounting for 15.4 and 14.4%, respectively. CONCLUSIONS Our results suggest that yaks with diarrhea serve as a reservoir of pathogenic E. coli carrying various virulence genes and resistance phenotypes. Therefore, clinicians and relevant authorities must ensure the regulatory use of antimicrobial agents and prevent the spread of these organisms through manure to farm workers and food-processing plants.