Aquatic biological monitoring is often used to inform and develop efficient conservation practices yet relies on conventional methods such as electrofishing, seining, visual surveys, etc. that are costly, time consuming, can miss detection of cryptic species, and induce organismal stress. Organismal stress and physiological status can be evaluated with biomarkers, such as epigenetic markers of DNA methylation (DNA-meth) and are indicative of population responses to environmental perturbations. Recent advances have shown methylation signatures are retained in shed DNA, lending environmental DNA (eDNA)- DNA that is present in environmental media -as a potential non-intrusive method of evaluating patterns of organismal physiology in conjunction with biodiversity. Here, we evaluate the utility of epigenetic-eDNA (epi-eDNA) in assessing DNA methylation patterns of juvenile Oncorhynchus mykiss, rainbow trout, responding to an acute 24-hour thermal challenge. By sequencing whole epigenetic genomes from fish and their respective tanks, we found 2328 loci that were differentially methylated between juveniles maintained in 75oC water and those maintained under 58oC, with tissue samples. With epi-eDNA sampling, we recovered many of these same sites, which retained these differential methylation patterns. These overlapping sites between tissue and epi-eDNA samples were found in genes corresponding to many physiological functions, including histone modifications, DNA damage responses, muscle physiology, cellular processes including membrane, ion transport and response to stress, and immune function. These results provide support that targeting epi-eDNA can uncover organismal physiological information in conjunction with biodiversity assessments, which may further the utility of eDNA studies in biomonitoring strategies.