The market demand for octopus grows each year, but landings are decreasing, and prices are rising. The present study investigated (1) diversity of Octopodidae in the Western Indian Ocean (WIO) and (2) connectivity and genetic structure of Octopus cyanea and O. vulgaris populations in order to obtain baseline data for management plans. A fragment of the cytochrome C oxidase subunit 1 (COI) gene was sequenced in 275 octopus individuals from Madagascar, Kenya and Tanzania. In addition, 41 sequences of O. vulgaris from South Africa, Brazil, Amsterdam Island, Tristan da Cunha, Senegal and Galicia were retrieved from databases and included in this study. Five different species were identified using DNA barcoding, with first records for O. oliveriand Callistoctopus luteus in the WIO. For O. cyanea (n = 229, 563 bp), 22 haplotypes were found, forming one haplogroup. AMOVA revealed shallow but significant genetic population structure among all sites (ϕST = 0.025, p = 0.02), with significant differentiation among: (1) Kanamai, (2) southern Kenya, Tanzania, North and West Madagascar, (3) Southwest Madagascar and (4) East Madagascar (ϕCT = 0.035, p = 0.017). For O. vulgaris (n = 71, 482 bp), 15 haplotypes were identified, forming three haplogroups. A significant genetic population structure was found among all sites (ϕST = 0.82, p ≤ 0.01). Based on pairwise ϕST-values and hierarchical AMOVAs, populations of O. vulgaris could be grouped as follows: (1) Brazil, (2) Madagascar and (3) all other sites. A significant increase in genetic distance with increasing geographic distance was found (Z = 232443, 81 r = 0.36, p = 0.039). These results indicate that for O. cyanea four regions should be considered as separate management units in the WIO. The very divergent haplogroups in O. vulgaris from Brazil and Madagascar might be evolving towards speciation and therefore should be considered as separate species in FAO statistics.