. Episodic floods and extended low or no flow periods characterise dryland river systems in Western Queensland, Australia. During protracted intervals between floods, rivers consist of a series of isolated waterholes, which serve as 'refugia' for aquatic species and much of the channel is dry. We categorised these waterholes into 'main waterholes', which are located in the main part of the river channel and 'satellite waterholes', which are located in distributary river channels. 2. We used mitochondrial sequences and allozymes to investigate levels of genetic diversity and patterns of connectivity among waterholes for two obligate freshwater species: Macrobrachium australiense (Decapoda: Palaemonidae) and Notopala sublineata (Gastropoda: Viviparidae). 3. We sampled 31 waterholes for M. australiense and 12 for N. sublineata. Based on a 505-bp fragment of cytochrome oxidase subunit I, we identified 54 haplotypes in a sample of 232 individuals for M. australiense and based on a 457-bp fragment of the same gene, 36 haplotypes in a sample of 145 individuals for N. sublineata. 4. Both nuclear and mitochondrial genetic data sets indicated that estimates of genetic diversity were not different in populations inhabiting main and satellite waterholes for either species. Also, there was generally very limited genetic differentiation among populations at any site. 5. We suggest that levels of connectivity among populations inhabiting waterholes at most sites are higher than expected. High levels of connectivity may help to maintain overall high levels of genetic diversity as well as low levels of genetic differentiation among waterholes within sites.