The goal of this study is to develop a new method to characterize taxonomically which microbes are present intracellularly in which cell types in the tumor microenvironment. The tumor microbiome impacts many aspects of tumor development including tumorigenesis, mutagenesis, response to chemotherapy and the immune response. A recent computational approach showed that bacterial reads found in sequencing tumor samples and taxon abundances are predictive of cancer type. Recent experimental evidence suggests that some bacteria in the tumor microbiome may reside intracellularly. We sought to answer two questions: 1) which microbes, especially bacteria, reside intracellularly in a tumor sample and 2) in which cell types do these microbes preferentially reside? We developed a computational pipeline, CSI-Microbes (computational identification of Cell-type-Specific Intracellular Microbes), to identify cell-type-specific intracellular microbes from single cell RNA-seq (scRNA-seq) data. CSI-Microbes builds on the observation that in contrast to extracellular microbes and contaminants, some cell-type-specific intracellular microbes would exhibit differential abundance between cell types. For validation, we show that CSI-Microbes identifies Salmonella as the only differentially abundant microbe in a scRNA-seq dataset in the subset of immune cells that were deliberately infected with Salmonella. Next, we recapitulate known findings that Merkel cell polyomavirus and Fusobacterium exist intracellularly in tumor cells in Merkel cell carcinoma and in colorectal carcinoma, respectively. We then apply CSI-Microbes to analyze scRNA-seq data from numerous cancer types. We find that Streptomyces is differentially abundant in the tumor cells of both breast and head-and-neck cancer. We identify three bacterial genera and four fungal genera that are differentially abundant specifically in the tumor cells of melanoma samples. We additionally find evidence for the reactivation of herpesvirus in the plasma cells of a patient with basal cell carcinoma after immune checkpoint blockade therapy. These sequence-based findings raise the possibility that these tumor-specific intracellular microbes may play functional roles in tumor metabolism and drug response in these cancer types. As such, they also may be a source of neo-antigens, which can potentially be targeted using T cell therapies. In sum, CSI-Microbes offers a new way for fast, single cell RNA-seq based identification of likely intracellular bacteria living within specific cell populations in tumors, markedly extending upon previous studies aimed at inferring microbial abundance from bulk tumor samples. Citation Format: Welles Robinson, Fiorella Schischlik, E. Michael Gertz, Alejandro A. Schäffer, Eytan Ruppin. Identifying the landscape of intratumoral microbes via a single cell transcriptomic analysis [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO093.