The sea lamprey (Petromyzon marinus), a non-native species in the Laurentian Great Lakes, has significantly impacted native fish communities and commercial fisheries, requiring population suppression efforts. While traditional control methods such as lampricides and barriers have reduced sea lamprey population abundance, questions remain regarding sea lamprey dietary composition given the focus of current damage assessments on economically and ecologically important host species. Recent advances in molecular technology offer promising methods of sea lamprey dietary assessment. Specifically, DNA metabarcoding enables species-specific identification of taxonomically diverse prey items from gut and fecal samples, and has proven effective in many taxa, including hematophagous species such as Arctic lamprey (Lethenteron camtschaticum) and sea lamprey. However, studies on DNA retention within digestive tracts are limited, particularly given the potential effects of environmental and dietary factors among hematophagous species. We used controlled feeding experiments to understand the effects these factors may have on DNA retention and host detectability within sea lamprey digestive tracts. Additionally, we evaluated the utility of metabarcoding for identifying multiple host species from consecutive feedings. Results indicate that host DNA can be detected up to 30 days post-feeding, with detection probability decreasing with increasing time following feeding. Temperature effects were dependent upon fasting periods, and host-switching trials indicated multiple previous host species could be detected from a single lamprey. Findings provide valuable insights for refining dietary analysis protocols for wild-caught sea lamprey within native and introduced ranges.