Freshwater ecosystems are particularly vulnerable to habitat loss and fragmentation, given the linear nature of rivers and streams. Many freshwater systems are affected worldwide, most notably by widespread construction of dams to meet increasing water demand for growing human populations. Small, fragmented populations of wildlife are vulnerable to stochastic events and loss of genetic diversity through drift, with an increased risk of inbreeding and the fixation of deleterious alleles. In addition, loss of genetic diversity reduces the adaptive potential of populations to respond to future environmental changes. By comparing microsatellite and hundreds of genome-wide markers, we investigated the impact of dams on neutral and adaptive genetic diversity and genetic structure of an endemic freshwater fish of Australia, Gadopsis marmoratus, in the Yarra River system (Victoria, Australia). A space-for-time design was implemented, with several populations sampled upstream and downstream in five streams systems of different size and with barriers of different known ages (from 45 to 120 years old). Results indicated a slight effect of barriers on genetic diversity in the smaller streams only. No effect of barrier age was detected. Indeed, catchment area and the presence of large reservoir upstream of barrier appear to prevent loss of genetic diversity upstream. Isolation-by-distance was more supported than Isolation-by-barrier in the whole study system. Our results, combined with computer simulations, provide general insights into the timing of barrier effects on different classes of molecular marker in freshwater systems.