Mesothelin targeting CAR T cells have limited activity in patients. In this study, we sought to determine if efficacy of anti-mesothelin CAR T cells is dependent on the mesothelin epitopes that are recognized by them. To do so, we developed hYP218 (against membrane-proximal epitope) and SS1 (against membrane-distal epitope) CAR T cells. Their efficacy was assessed in vitro using mesothelin positive tumor cell lines and in vivo in NSG mice with mesothelin expressing ovarian cancer (OVCAR-8), pancreatic cancer (KLM-1) and mesothelioma patient-derived (NCI-Meso63) tumor xenografts. Persistence and tumor infiltration of CAR T cells was determined using flow cytometry. hYP218 CAR T cells killed cancer cells more efficiently than SS1 CAR T cells, with 2-4-fold lower ET50 value (Effector to Target ratio for 50% killing of tumor cells). In mice with established tumors, single intravenous administration of hYP218 CAR T cells lead to improved tumor response and survival compared to SS1 CAR T cells, with complete regression of OVCAR-8 and NCI-Meso63 tumors. Compared to SS1 CAR T cells, there was increased peripheral blood expansion, persistence, and tumor infiltration of hYP218 CAR T cells in the KLM-1 tumor model. Persistence of hYP218 CAR T cells in treated mice led to anti-tumor immunity when rechallenged with KLM-1 tumor cells. Our results demonstrate that hYP218 CAR T cells, targeting mesothelin epitope close to cell membrane, are very effective against mesothelin positive tumors and are associated with increased persistence and tumor infiltration. These results support its clinical development to treat patients with mesothelin expressing cancers.