Intrapopulation variation in personality, including activity, boldness, and aggressiveness, is becoming more widely recognised and is hypothesised to substantially affect ecological and evolutionary dynamics. Although previous studies utilised candidate-gene approaches and genome-wide association analyses to identify genes correlated with variations in activity and aggressiveness, personality variation may not be fully captured in the nuclear genome, as it does not account for mitochondrial genomes. Mitochondrial genes encode products that are key regulators of the cellular energy-producing pathways in metabolic processes and are thought to play a significant role in life history and reproductive traits. In this study, we considered many isofemale lines of Drosophila immigrans established from two wild populations to investigate whether intrapopulation variation in the mitochondrial genome affected activity level within this species. We identified two major haplogroups in these populations, and activity levels in both larvae and adults differed significantly between the two haplogroups. This result indicated that intrapopulation activity level may be partially controlled by mitochondrial genes, along with the interaction between nuclear and mitochondrial genes and the age of individual organisms.