MADS-box transcription factors are known to be involved in many important processes during plant growth and development. To date, few cucumber MADS-box genes and little tissue expression profiling have been reported. Recent completion of the cucumber whole-genome sequencing has allowed genome-wide analysis of the MADS-box gene family in cucumber as well as its comparison with other species. Here, we performed comprehensive analyses of the 43 cucumber MADS-box genes and compared them with those in Arabidopsis, poplar, and grapevine. The phylogenetic analysis showed that most cucumber members were comparable with those in other species, with the exception of AG members. At the same time, the three subfamilies FLC, AGL12, and Bs were absent in the cucumber genome. The conserved motif analysis revealed that most motifs outside the MADS domain were distributed only in specific groups. The analysis of chromosomal localization suggested that tandem duplication might contribute to the MADS-box gene expansion. Expression analysis revealed that 42 of 43 cucumber MADS-box members were expressed in multiple plant tissues, thereby implying their various roles in plants.