The Darling hardyhead, Craterocephalus amniculus (Atherinidae), is a threatened fish species inhabiting upstream reaches of a number of northern Murray-Darling Basin catchments. We examined patterns of seasonal size structure, interannual and spatial variation in diet, and habitat selection in this species across three sites and four seasons in the upper Macintyre River, northern New South Wales. Size structure data suggested a single annual spawning season in late September or early October, with only a single cohort apparent in the population in three of the four seasons. Dietary diversity increased with distance downstream. At the two upstream sites, aquatic invertebrates made up most of the diet while over half the gut contents at the downstream site was unidentified detritus. Diets varied significantly, both between seasons at the downstream site and among the three sites. Preference was shown for pool habitats with a sand or cobble substrate, increased channel depth and width and distance from the bank, and reduced flow velocity, while in-stream woody debris cover, and exotic riparian vegetation cover were avoided. Darling hardyhead may be vulnerable to further population decline in light of its narrow spawning season and habitat preferences. However, comparable data from nearby catchments will be necessary to ascertain the species’ conservation status across its broader distribution.