BACKGROUND Accurately reporting the identity and representation of enteric nervous system (ENS) neuronal subtypes along the length of the gastrointestinal (GI) tract is critical to advancing our understanding of ENS control of GI function. Reports of varying proportions of subtype marker expression have employed different dissection techniques to achieve wholemount muscularis preparations of myenteric plexus. In this study, we asked whether differences in GI dissection methods could introduce variability into the quantification of marker expression. METHODS We compared three commonly used methods of ENS wholemount dissection: two flat-sheet preparations that differed in the order of microdissection and fixation and a third rod-mounted peeling technique. We also tested a reversed orientation variation of flat-sheet peeling, two step-by-step variations of the rod peeling technique, and whole-gut fixation as a tube. We assessed marker expression using immunohistochemistry, genetic reporter lines, confocal microscopy, and automated image analysis. CONCLUSIONS We found no significant differences between the two flat-sheet preparation methods in the expression of calretinin or neuronal nitric oxide synthase (nNOS) as a proportion of total neurons in ileum myenteric plexus. However, the rod-mounted peeling method resulted in decreased proportion of neurons labeled for both calretinin and nNOS. This method also resulted in decreased transgenic reporter fluorescent protein (tdTomato) for substance P in distal colon and choline acetyltransferase (ChAT) in both ileum and distal colon. These results suggest that labeling among some markers, both native protein and transgenic fluorescent reporters, is decreased by the rod-mounted mechanical method of peeling. The step-by-step variations of this method point to mechanical manipulation of the tissue as the likely cause of decreased labeling. Our study thereby demonstrates a critical variability in wholemount muscularis dissection methods.