Euseius nicholsi (Ehara et Lee) is an important indigenous predator of many species of pest mites and insects in China. In this study, the genetic diversity of Euseius nicholsi among ten populations was investigated using coxI and ITS sequences from 145 individuals. This indicated the existence of 33 and 16 haplotypes, respectively. Most of the populations studied showed high levels of haplotype and nucleotide diversity. In the coxI region, haplotype and nucleotide diversity varied among populations from 0.385 to 0.809 and from 0.00342 to 0.01111, respectively. In the ITS region they varied from 0.000 to 0.803 and from 0.00000 to 0.00185, respectively. For both gene regions, the highest haplotype and nucleotide diversities were detected in Chengdu (CD) and Chongqing (CQ) from southwestern China. The results of population differentiation suggested that E. nicholsi indicated at least three geographically approximate populations, the middle and lower reaches of the Yangtze River (WH, CS, JJ, NP, and NJ), southwestern China (CQ and CD), and southern China (NN, GZ, and SG). High levels of genetic diversity were observed in most populations. This study serves as an introductory probe into the genetic differentiation of E. nicholsi, for which little molecular information is available. Other genetic markers and intensive sampling in the following studies would be useful and necessary for more detailed classifications of this taxon.