The increasing prevalence of antimicrobial resistance has raised significant concern globally. Colistin is currently considered a last resort for treating Gram-negative bacterial infections. However, the emergence of colistin resistance has led to a difficult situation against bacterial infections. Therefore, the monitoring of colistin resistance is of great importance for the control of bacterial infections. The mobile colistin resistance (mcr) gene has been identified as a colistin resistance gene, and ten mcr genes (mcr-1 to mcr-10) have been identified to date. Hence, the detection of mcr genes can help predict bacterial colistin resistance at the molecular level. However, there have not been reported multiplex PCR methods for simultaneously detecting mcr-1 to mcr-10 until now. In this study, we established a one-step multiplex PCR method for simultaneous detection of mcr-1 to mcr-10 for the first time. Furthermore, we retrospectively investigated the prevalence of the ten mcr genes in Escherichia coli (E. coli) and Salmonella isolates in China. The results showed that the mcr detection rate of Salmonella isolated during 2004-2019 was 4.73% (16/338), and only mcr-9 was harbored. As well, the mcr detection rate of E. coli isolated during 2012-2015 was 20.42% (49/240) and only mcr-1 was identified. Moreover, we also investigated the relationship between mcr harboring and colistin phenotype-resistance. The broth micro-dilution assay results showed that all mcr-1-positive E. coli isolates were colistin-resistant. However, all mcr-9-positive Salmonella isolates did not represent colistin-resistance. Our findings are beneficial for the monitoring and control of colistin resistance.