Understanding the significance of a river reach to a particular species is critical for informing riverine restoration and management. Generally, the relative significance of a river reach for freshwater turtles is based upon population counts. However, capture rates can be greatly influenced by the methods employed, species behavior, localized in-stream conditions, and the operator's knowledge and skill. Here, we report on abundance shifts within freshwater turtle assemblages along a river continuum using a protocol that standardized the sampling effort. Turtle capture was undertaken at 20 study sites along the Mary River (Queensland, Australia), and repeated identically every 6 mo over a 2-yr period. A large funnel trap with a 30-m wingspan was deployed at each site and turtles were captured over a 4-d period. The turtle species assemblages significantly differed between the upper, mid, and lower catchments (multivariate analysis of variance, p < 0.05), suggesting species preference for the broad geomorphological and ecological features of each reach. The observed spatial variance in species assemblage was consistent over time and unaffected by the season, demonstrating that the assessment was repeatable and unaffected by individual species' life history patterns. We argue that trends in turtle species assemblage could be used instead of absolute species count data to alert natural resource managers to shifts in conditions and provide early warning signs of habitat degradation or management success. The technique is cheaper and easier to implement than abundance counts and ensures that capture biases remain constant under different conditions and operators.