Solanum lycopersicoides is a valuable genetic resource for tomato (Lycopersicon esculentum) genetic improvement. However, there are few reports on its agronomic traits such as disease resistance and cold tolerance. In this paper, the resistance to cucumber mosaic virus (CMV) and leaf mold (Cladosporium fulvum Cooke) and cold tolerance of five lines of S. lycopersicoides were studied through investigation of disease inoculation and electrolyte leakage analysis. The results showed that S. lycopersicoides was highly resistant or immune to CMV and leaf mold and more tolerant to low temperature than L. esculentum. This study is helpful for the genetic improvement of tomato by using S. lycopersicoides as breeding materials.