A new infestation of the Formosan subterranean termite, Coptotermes formosanus Shiraki (Blattodea: Rhinotermitidae), was discovered in Canyon Lake, Riverside County, California. We used three mitochondrial DNA (COI, COII, and 16S) and seven polymorphic microsatellite markers to characterize the genetic relationship of the colony with two other colonies that were collected in 1992 and 2018 in La Mesa, San Diego County. Maximum likelihood phylogeny of C. formosanus based on concatenated COI and COII sequences revealed that the two La Mesa populations (CA01 and CA02) and the Canyon Lake population (CA03) were from different maternal lineages. Based on the 14 COII haplotypes of C. formosanus found world-wide, CA01 and CA02 belonged to a haplotype widely distributed across the United States, while CA03 was grouped under a haplotype predominantly found in Asia. Microsatellite allele frequencies across all loci for both La Mesa populations were relatively similar, but significant genetic differences were found between CA02 and CA03 colonies (F_ST = 0.24; Dest = 0.30; G″_ST = 0.55; P < 0.01).