Spatial and temporal distribution data provide critical information for invasive species management. For example, distribution data can help managers with early detections and guiding other response actions. Environmental DNA (eDNA)-based sampling exists as one tool for monitoring invasive species. As part of bigheaded carp Hypophthalmichthys spp. monitoring efforts in the Illinois River, USA, we compared eDNA-based sampling results at multiple habitats across an invasion gradient in 2015. Greater densities of carp occurred downriver in the Illinois River and lower densities occurred upriver. We sampled from five locations along this gradient and from three habitat types (backwater, main channel, and shoreline) within each location. We sampled each location in April and June. A priori, we hypothesized that more eDNA detections would occur downriver, where higher densities of carp occur; that more eDNA detections would occur in backwater habitats compared to areas of the river with more fish movement; and that more eDNA detections would occur in April, because bigheaded carps are thought to use our sampling areas more during the spring. We compared the proportion of samples positive across this gradient, the habitat type, and the two sampling time periods. The most downriver location had the highest proportion of samples with eDNA detections, the backwater habitats had the highest proportion of samples with eDNA detections, and April had more positive detections than June. Our results highlight the importance of sampling across multiple habitat types and across time to gain a clear understanding of distribution when using eDNA-based sampling. Thus, being cognizant of the interactions between seasonal habitat use and eDNA-based detections is important for managers who rely upon eDNA-based monitoring.