Plants confront with different environmental and abiotic stress stimuli such as cold, drought and salinity, which affect growth and development and limits plant productivity and yield. Plants physiological and biochemical pathways respond to overcome stress challenges. Calcium(Ca2+) has emerged as a significant secondary messenger that regulates the activities of hormonal and environmental signals that are associated with biotic and abiotic stresses. Ca2+ binding proteins typically contain a Ca2+ binding EF-hand(a helix-loop-helix structure) motif. Calmodulin-like(CML) proteins are an important Ca2+ sensor and play a significant role in the mediating plant stress responsive mechanism.In this study, tomato genes encoding calmodulin-like(CML) proteins that possess EF-hand motifs, and no other identifiable functional domains were analyzed. Using genome analysis and BLAST searches in the database, 52 CML genes were identified in tomato. Comprehensive analyzes, including evolutionary relationships, gene structures, chromosomal locations, functional annotations, and gene duplications, were performed. Distribution mapping exhibited that 52 Sl CML genes containing different intron/exon patterns were unevenly distributed among ten chromosomes. In addition, 24 Sl CML proteins were predicted as segmentally duplicated. Conserved motifs, promoter cisregulatory elements, organ-based expression patterns and expression analyzes indicated the potential responsiveness of Sl CML proteins to abiotic stresses and phytohormones. These results illustrate the complexity of the CML gene family and indicate a potentially vital role for these molecules in tomato growth and development as Ca2+ signal transducers.Furthermore, cold responsive calmodulin-like(Sh CML44) gene was isolated from cold tolerant wild tomato(Solanum habrochaites), and functionally characterized. The Sh CML44 was differentially expressed in all plant tissues including root, stem, leaf, flower and fruit, and was strongly up-regulated under cold, drought and salinity stresses along with plant growth hormones. Under cold stress, progressive increase in the expression of Sh CML44 was observed particularly in cold-tolerant S. habrochaites. The Sh CML44-overexpressed plants showed greater tolerance to cold, drought, and salinity stresses, and improved germination and better seedling growth. Transgenic tomato plants demonstrated higher antioxidant enzymes activity, gas exchange and water retention capacity with lower malondialdehyde accumulation and membrane damage under cold and drought stresses compared to wild-type. Moreover, transgenic plants exhibited reduced reactive oxygen species and higher relative water contents under cold and drought stress, respectively. Greater stress tolerance of transgenic plants was further reflected by the up-/down-regulation of stress-related genes including SOD, GST, CAT, POD, LOX, PR and ERD. In crux, these results strengthen the molecular understanding of Sh CML44 gene to improve the abiotic stress tolerance in tomato.