Mitogen-activated protein kinases (MAPKs), a family of Ser/Thr protein kinases, play an essential role in mediating biotic and abiotic stress responses in plants. In this study, we investigated 16 putative SlMAPK genes from tomato genome and compared them with those from Arabidopsis. The full cDNA sequences of 13 novel SlMAPKs in tomato were obtained through PCR ampilification. A comprehensive genome-wide analysis of SlMAPKs in tomato is presented, including their gene structure, phylogeny, genome localization, and expression profiles. Phylogenic analysis of the 16 SlMAPKs and 20 AtMAPKs from Arabidopsis indicated that the SlMAPK genes were clustered into four major groups, and genes within the same groups had similar exon-intron structures. All SlMAPK proteins in groups A, B and C had a Thr-Glu-Tyr (TEY) activation domain, whereas those in group D contained a Thr-Asp-Tyr (TDY) activation domain. The analysis of 5'-upstream region of SlMAPKs revealed a group of putative cis-acting elements related to stress responses. Expression analysis of SlMAPK genes using RT-PCR and real-time quantitative PCR demonstrated that all SlMAPK transcripts were able to be detected in at least one investigated tissue, and some of them exhibited tissue-specific expression patterns. The transcript abundance of nearly all SlMAPK genes was increased in response to heat stress treatment. Our data provided an insight into the evolution of the gene family and a useful reference for further functional analysis of MAPK family genes in tomato.