Freshwater organisms generally exhibit strong genetic structuring. Although understanding the level and spatial distribution of genetic diversity is crucial for conservation management planning, such information has received little attention until recently in highly developed cities where local extinction attributed to habitat destruction and urbanisation is intense. We compared the genetic divergence in mitochondrial COI of the freshwater shrimp Caridina cantonensis collected from 32 sites in Hong Kong, to determine the connectivity among populations and the levels of genetic diversity of the shrimp. We found that shrimp from different streams are genetically highly differentiated and each stream always has its own unique haplotype groups, even though some of the streams are separated only by a few kilometres, indicating very limited gene flow across streams. Moreover, genetic diversity within each stream is very low, usually with a single haplotype dominating the entire population, and genetic differentiation was observed among tributaries from the same drainage. The high genetic diversity of these species over short distances has significant conservation implications because a substantial amount of biodiversity may have already been lost as a result of past development. Careful conservation planning is essential for future development in Hong Kong and other cities.