Histone modifications affect gene expression, but the mechanism and biological consequence of natural variation in histone modifications remain unclear. Here, we generated genome-wide integrated maps of H3K27me3 modification and transcriptome for Col, C24 and their F1 hybrid. A total of 1,828 genomic regions showing variation in H3K27me3 modification between Col and C24 were identified, most of which were associated with genic regions. Natural variation of H3K27me3 modification between parents could result in allelic bias of H3K27me3 in hybrids. Furthermore, we found that H3K27me3 variation between Col and C24 was negatively correlated with gene expression differences between two accessions, especially with those arising from the cis-effect. Importantly, mutation of CLF, an Arabidopsis methyltransferase for H3K27, altered gene expression patterns between the parents. Together, these data provide insights into natural variation of histone modifications and their association with gene expression differences between Arabidopsis ecotypes.