AbstractA variety of methods have been developed to demultiplex pooled samples in a single cell RNA sequencing (scRNA-seq) experiment which either require hashtag barcodes or sample genotypes prior to pooling. We introduce scSplit which utilizes genetic differences inferred from scRNA-seq data alone to demultiplex pooled samples. scSplit also enables mapping clusters to original samples. Using simulated, merged, and pooled multi-individual datasets, we show that scSplit prediction is highly concordant with demuxlet predictions and is highly consistent with the known truth in cell-hashing dataset. scSplit is ideally suited to samples without external genotype information and is available at: https://github.com/jon-xu/scSplit