Objective　This study aimed to analyze the mitochondrial cytochrome c oxidase subunit I (mtDNA-CO Ⅰ) gene in Aedes albopictus populations from Ji'nan and Ji'ning Cities in Shandong Province, in order to reveal the genetic differences and phylogenetic relationships among different subpopulations and to investigate the relationship between genetic diversity and geographical distribution of Aedes albopictus. Method　In this study, Ae. Albopictus mosquitoes were collected from Licheng District in Ji'nan City and Rencheng District in Ji'ning City. The sequences of mtDNA-CO Ⅰ gene of Ae. albopictus populations was amplified by PCR, and the results were aligned and compared using the BLAST tool from NCBI. Data analysis was then conducted using the software of BioEdit 7.0, DnaSP v6 and PopART 1.7. Genetic diversity parameters for Aedes albopictus, and a haplotype network was constructed. Results　Gene sequencing of the mtDNA-CO Ⅰ gene in Aedes albopictus populations from Ji'nan and Ji'ning cities in Shandong Province resulted in 86 and 84 gene fragments, respectively. BLAST comparison revealed five mutation sites in all the sequences, with a nucleotide diversity (Pi) of 0.000 97 and a G+C content of 33.4%, reflecting the characteristics of mitochondrial DNA. Compared to Ji'ning, the Ae. albopictus population in Ji'nan exhibited higher nucleotide diversity and haplotype diversity, as well as greater nucleotide differences, though these differences were not statistically significant. Furthermore, both cities had six haplotypes of Aedes albopictus, with three shared haplotypes and three unique haplotypes found only in the population of Ji'nan City. Population structure analysis revealed minimal nucleotide differences between Ae. albopictus populations of Ji'nan and Ji'ning. Conclusions　The mtDNA-CO Ⅰ gene can be used as a molecular marker for species identification and for studying genetic diversity of Ae. albopictus populations, thereby providing support for more effective classification and control strategies.