We previously demonstrated that allopolyploidization could induce phenotypic variations and genome changes in a newly synthesized allotetraploid in Cucumis. To explore the molecular involvement of epigenetic phenomena, we investigated cytosine methylation in Cucumis by using methylation-sensitive amplified polymorphism (MSAP). Results revealed a twofold difference in the level of cytosine methylation between the reciprocal F1 hybrids and the allotetraploid. Analysis of the methylation pattern indicated that methylation changed at 2.0% to 6.4% of total sites in both the F1 hybrids and the allotetraploid compared with their corresponding parents. Furthermore, 68.2% to 80.0% of the changed sites showed an increase in cytosine methylation and a majority of the methylated sites were from the maternal parent. Observations in different generations of the allotetraploid found that the extent of change in cytosine methylation pattern between the S1 and S2 was significantly higher than that between the S2 and S3, suggesting stability in advanced generations. Analysis of 7 altered sequences indicated their similarity to known functional genes or genes involved in regulating gene expression. Reverse transcription - polymerase chain reaction analysis suggested that at least two of the methylation changes might be related to gene expression changes, which further supports the hypothesis that DNA methylation plays a significant role in allopolyploidization.