We analyzed a portion of mitochondrial COI gene sequences (406 bp) to investigate the genetic diversity and geographic variation of the mushroom pest flies Lycoriella mali and Coboldia fuscipes in Korea. L. mali showed minimal sequence divergence (0.2%) in two mtDNA haplotypes, whereas C. fuscipes showed an intermediate level of sequence divergence (1.2% at maximum) compared with other relevant studies. While L. mali was fixed with one haplotype except for one population, C. fuscipes possessed a total of ten mtDNA haplotypes, and six of these occurred commonly in multiple populations. We ascribed the difference in the level of genetic variation between the two species to a difference in the degree to which they are dependent on cultured mushroom, which is a fluctuating food source. In C. fuscipes, as in other cosmopolitan insect pest species, a high rate of female migration (Nm=0.947–infinite) and little genetic differentiation (FST=0.345–−0.094) between populations were estimated.