Esophageal squamous cell carcinoma (ESCC) has a high incidence, poor treatment response, and high mortality. Subcutaneous transplant tumor models are commonly used to study the immunosuppressive tumor microenvironment and its impact on immunotherapy. In this study, we established two new ESCC mouse cell lines, mEC525M and mEC586F, from 4NQO-induced ESCC mouse models of different sexes. We compared their proliferation, motility, and molecular characteristics with existing lines (HNM007, AKR, mEC25) using in vitro experiments and whole-exome sequencing. Treatment sensitivity analysis of all murine ESCC tumor cell lines revealed that AKR and HNM007 cells were more responsive to chemotherapy, mEC25 cells were more sensitive to radiotherapy, whereas mEC525M and mEC586F cells exhibited greater sensitivity to immunotherapy. Multiplex immunohistochemistry (mIHC) staining analysis revealed differences in immune infiltration among the tumors derived from the five mouse ESCC cell lines, with the highest proportion of T cells in mEC525M tumors, the highest proportion of CD11b