We compared serotypes, drug susceptibility and presence of virulence-related genes in diarrhoeagenic Escherichia coli isolates from children < 5 years from Kenya (n = 82) and Japan (n = 47). Multiplex PCR was used to detect genes coding for enteroaggregative adherence (aggR), heat-stable toxin (st), heat-labile toxin (It), verotoxin (vt), attaching and effacing mechanism (eaeA), enteroaggregative E. coli heat-stable enterotoxin 1 (astA) and enteroinvasive mechanism (invE). Kenyan E. coli O-serotypes were more diverse than those from Japan (29 vs. 12 serotypes) and exhibited high level multidrug resistance to World Health Organization (WHO) recommended antibiotics. Resistance rates to tetracycline, ampicillin and sulphamethoxazole-trimethoprim were 70.7, 65.9 and 68.3% respectively, but resistance to sulphamethoxazole-trimethoprim among the E. coli isolates from Japan was low (21%). Kenyan isolates harboured virulence-related genes in high frequency (82.9%) compared to those from Japan (25.5%) with aggR and astA being the most frequently detected genes. The presence of multiple virulence genes was associated with multidrug resistance and this merits further investigation.