Aims: This research examined the genetic structure of the Mudskipper Boleophthalmus dussumieri across three key intertidal zones in the north of the Persian Gulf and Oman Sea, Iran.
Materials & Methods: Genetic analysis of the mitochondrial cytochrome b gene in 28 samples (25 contemporary and 3 archived) from four regions evaluated diversity and structure using haplotype and nucleotide diversity indices, AMOVA, ΦST, and haplotype networks.
Finding: A total of 21 haplotypes were identified from 28 individuals. Overall haplotype diversity was high (Hd = 0.942), and nucleotide diversity was moderate (π = 0.013). A hierarchical analysis of molecular variance (AMOVA) revealed significant genetic structuring among the three B. dussumieri populations surveyed. The analysis indicated that 27.87% of the total genetic variation was partitioned among populations, while the remaining 72.16% was found within populations. The overall fixation index (ΦST) was 0.72, a statistically significant value (p < 0.001), confirming high and definitive genetic differentiation across the sampling region. 
Conclusion: The results show a clear population-genetic pattern, marked by distinct differences between the Goban and Makran populations. In contrast, the Delvar and Khorabi populations form a single, genetically uniform cluster. These patterns align with a model of recent demographic expansion from a common ancestral source, followed by initial genetic divergence. This differentiation is influenced by current barriers to gene flow and the species’ limited dispersal ability.