Adult specimens of Panulirus penicillatus were collected from localities distributed in Japan: Hachijojima Island, Ryukyu Archipelago (Amamioshima, Okinawajima, Ishigakijima); Taiwan: Taitung; Indonesia: Java Sea and Gebe Island; French Polynesia: Moorea Island; and Ecuador: Isabela Island. Phyllosoma larval specimens were collected from the western sea of the Galápagos Islands. In total, 569–570 nucleotide sequences were analyzed from the mtDNA control region of 480 individuals. Almost all individuals had a unique haplotype. Intra-population haplotype (h) and nucleotide (π) diversities were high for each locality, ranging h = 0.9996-1.0000 and π = 0.0255-0.0448. A mismatch distribution of pairwise differences between haplotypes indicated that P. penicillatus does not fit the sudden population expansion model. Analysis of molecular variance (AMOVA) shows a clear genetic difference between two regions (western/central Pacific and eastern Pacific). High gene flow was found within localities in the western/central Pacific region, probably resulting from an extended planktonic larval stage and prevailing ocean currents. In this study we detected genetic structure between eastern Pacific populations and western Pacific populations, indicating that despite the nearly 1-yr larval period for this species, larvae generally do not pass over the East Pacific Barrier.