Cathepsin B-like protease 2 (CathB2) genes have been shown to participate in programmed cell death (PCD) that is a crucial component of development and defense mechanisms in plants at biotic and abiotic stresses. However, little is known regarding CathB2 genes in tomato. In this study, we reported the analysis of two SlCathB2 genes named SlCathB2−1 and SlCathB2−2. Promoter analysis found some elements related to abiotic stress-responses in two SlCathB2 genes. Domain analysis revealed that SlCathB2 encoding proteins characterized by signal peptide, propeptide, and papain family cysteine protease domain. The phylogenetic analysis revealed their close evolutionary relationship with the CathB2 of Solanum tuberosum. Expression analysis showed that SlCathB2 differentially expressed in various tissues and SlCathB2 positively responded to high temperature, drought, salt, ABA and SA treatments with higher expression upon high-temperature stress (HS) in the cultivated tomato line ‘LA1698′ (Solanum lycopersicum) and the currant tomato line ‘LA2093′ (Solanum pimpinellifolium). Then, the expression of SlCathB2 in the leaves of tomato genotypes with different thermotolerance under moderately elevated temperature (MET, 33 °C/33 °C) and acutely elevated temperature (AET, 40 °C/40 °C) stresses were further analyzed. The results showed that SlCathB2 genes were most triggered in heat-sensitive tomato genotypes (S1 and S2) as compared to heat-tolerant genotypes (T1 and T2) and were not significantly induced in the moderate genotypes (M1 and M2) under MET and AET stresses, suggesting that they were negatively regulated by HS. Furthermore, SlCathB2 genes showed similar expression patterns in tomato genotypes with same behavior for thermotolerance under MET and AET stresses, while exhibited higher expression level under AET stress, indicating that more thermosensors and multiple HSR (high-temperature stress- responsive) pathways were activated under AET stress leading to the higher expression level of a set of HSR genes including SlCathB2−1 and SlCathB2−2 genes. This study lays a foundation for functional studies of SlCathB2 in tomato.