The trabecular meshwork within the outflow apparatus is critical in maintaining intraocular pressure homeostasis. In vitro studies employing primary cell cultures of the human trabecular meshwork (hTM) have conventionally served as surrogates for investigating the pathobiology of TM dysfunction. Despite its abundant use, translation of outcomes from in vitro studies to ex vivo and/or in vivo studies remains a challenge. Given the cell heterogeneity, performing single-cell RNA sequencing comparing primary hTM cell cultures to hTM tissue may provide important insights on cellular identity and translatability, as such an approach has not been reported before. In this study, we assembled a total of 14 primary hTM in vitro samples across passages 1-4, including 4 samples from individuals diagnosed with glaucoma. This dataset offers a comprehensive transcriptomic resource of primary hTM in vitro scRNA-seq data to study global changes in gene expression in comparison to cells in tissue in situ. We have performed extensive preprocessing and quality control, allowing the research community to access and utilize this public resource.