Aquatic invasive species (AIS) are a threat to freshwater ecosystems. Understanding AIS prevalence is critical to effective management and early detections. However, traditional monitoring for AIS is time and resource intensive. Environmental DNA (eDNA) has the potential to alleviate key issues related to traditional monitoring, but questions persist about how eDNA detection probability can be optimized across multiple species with different life histories. To quantify spatio-temporal variation in detection probability of AIS using eDNA sampling, we sampled 21 lakes with known populations of invasive common carp, rusty crayfish, spiny water flea, and zebra mussels 5 times over the course of the open water season. We used qPCR with species-specific assays to determine presence of each species in water samples. With Bayesian occupancy models, we quantified the effects of lake and site characteristics and sampling season on eDNA detection probability. Our results suggest that species-specific probability of detection varies seasonally and is related to the species’ life history. Our results will provide guidance to organizations interested in using eDNA as a monitoring tool.