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¹ Conservation Biology Research Group, Department of Biology, University of Malta, Msida MSD2080, Malta DOI : 10.4194/2459-1831-v4_1_05 Viewed : 4031 - Downloaded : 2651 The freshwater crab, Potamon fluviatile inhabits a number of watercourses in Italy, the Balkan Peninsula and some neighbouring islands. In this study, P. fluviatile specimens (n = 176) were tissue sampled from different watercourses in Maltese archipelago and were genetically analysed to investigate the population structure of the species within this small geographical area. The DNA sequences analysed included 622 nucleotides from cytochrome oxidase c subunit 1 gene, and 3160 nucleotides from the sequence between the NADH dehydrogenase subunit 6 gene and the 16S rRNA gene. Results indicated the occurrence of two main clades, with most populations exhibiting different haplotype frequencies and were characterized by private haplotypes that radiate from the main haplotype of each clade. This led to significant pairwise differences between most of the sampled populations. On grouping populations based on their geographical region, it was found that there are significantly large genetic differences between regions, with AMOVA analyses showing that over 90% of the genetic variation is due to differences between distant water systems. The population structure and the lack of genetic connectivity found among populations inhabiting different valleys highlight the urgent need for targeted conservation management of this legally protected species that is fragmented in small isolated populations. Keywords : Maltese archipelago, Population structure, Freshwater courses, mtDNA, Genetic connectivity