AbstractEnvironmental DNA (eDNA) has begun to show promise as a robust and reproducible tool for monitoring cetaceans in coastal and offshore waters. Some limiting factors preventing the wider application of eDNA for cetacean monitoring includes lack of species‐specific qPCR assays and limited in situ validation. In this study, we determined 15 monitoring stations within cetacean hotspots in Chatham Sound (British Columbia, Canada), from which we collected a combination of visual and acoustic data, and low‐volume eDNA samples (equivalent to ~250 mL seawater). We designed novel eDNA assays for gray whale and Dall's porpoise and validated existing assays for harbor porpoise, killer whale, and humpback whale. Overall, we collected a total of 120 paired eDNA samples across four sampling intervals, 60 preserved with absolute ethanol and 60 preserved with propylene glycol antifreeze. Positive rates for visual (18%) and acoustic (4%) detections were higher than the eDNA detection rate (<3%), with only one sample (antifreeze‐preserved) producing a positive detection for humpback whales at one of the stations. We discuss factors which could have influenced the lack of detections and highlight the need for higher sample volumes and species‐specific sample approaches to improve detection success and confidence in eDNA applicability for cetacean monitoring.