Escherichia coli isolated from colon, cecal, and fecal samples are commonly used as indicator organisms to monitor antimicrobial resistance (AMR) in wild animals. The effect of sample type on E. coli recovery and AMR detection is unknown. We compared the prevalence of E. coli, the frequency of AMR, and the level of agreement between cecal, colon, and fecal samples collected from individual wild house mice (Mus musculus). Samples were collected from 49 mice trapped at swine farms, May–October 2008. We found no difference in the proportion of E. coli– positive samples (82%) among sample types and the agreement among sample types for E. coli recovery ranged from moderate to almost perfect. The percentage of E. coli positive samples resistant to one or more antimicrobial varied from 53% for colon samples to 71% for cecum samples; however, there was no significant difference in the proportion of resistant samples among sample types. The agreement among sample types for resistance to one or more antimicrobial ranged from fair to substantial. These findings indicate that there is no definitive sample type for studies of AMR in house mice. However, we suggest that fecal samples, which have direct contact with the environment, are likely the best sample to use in studies assessing the potential impact of AMR in wildlife on environmental and public health.