Abstract Summary Subpathways, which are defined as local gene subregions within a biological pathway, have been reported to be associated with the occurrence and development of cancer. The recent subpathway identification tools generally identify differentially expressed subpathways between normal and cancer samples. psSubpathway is a novel systems biology R-based software package that enables flexible identification of phenotype-specific subpathways in a cancer dataset with multiple categories (such as multiple subtypes and developmental stages of cancer). The operation modes include extraction of subpathways from pathway networks, inference with subpathway activities in the context of gene expression data, identification of subtype-specific subpathways, identification of dynamic-changed subpathways associated with the cancer developmental stage and visualization of subpathway activities of samples in different phenotypes. Its capabilities enable psSubpathway to find specific abnormal subpathways in the datasets with multi-phenotype categories and to fill the gaps in the recent tools. psSubpathway may identify more specific biomarkers to facilitate the development of tailored treatment for patients with cancer. Availability and implementation The package is implemented in R and available under GPL-2 license from the CRAN website (https://cran.r-project.org/web/packages/psSubpathway/). Supplementary information Supplementary data are available at Bioinformatics online.