Sugarcane mosaic disease, mainly caused by Sugarcane streak mosaic virus (SCSMV), has serious adverse effects on the yield and quality of sugarcane. Eukaryotic translation initiation factor 4E (eIF4E) is a natural resistance gene in plants. The eIF4E-mediated natural recessive resistance results from non-synonymous mutations of the eIF4E protein. In this study, two sugarcane varieties, CP94-1100 and ROC22, were selected for analysis of their differences in resistance to SCSMV. Four-base missense mutations in the ORF region of eIF4E resulted in different conserved domains. Therefore, the differences in resistance to SCSMV are due to the inherent differences in eIF4E of the sugarcane varieties. The coding regions of eIF4E included 28 SNP loci and no InDel loci, which were affected by negative selection and were relatively conserved. A total of 11 haploids encoded 11 protein sequences. Prediction of the protein spatial structure revealed three non-synonymous mutation sites for amino acids located in the cap pocket of eIF4E; one of these sites existed only in a resistant material (Yuetang 55), whereas the other site existed only in a susceptible material (ROC22), suggesting that these two sites might be related to the resistance to SCSMV. The results provide a strong basis for further analysis of the functional role of eIF4E in regulating mosaic resistance in sugarcane.