Studies have shown that plant flowering time is affected by endogenous and exogenous factors, but the role of DNA methylation in flowering still remains unclear. Here, 27 Arabidopsis thaliana populations were used to determine how autonomous pathway gene methylation affects flowering differences by regulating gene expression patterns. DNA methylation analysis, qPCR and transgenic verification were performed. The flowering time of the Arabidopsis populations ranged from 19 to 55 days. Methylation of the coding regions of six upstream genes in the autonomous pathway, FVE, FY, FLD, PEP, HDA5 and PPR 39-1, was significantly correlated with flowering time and relative expression levels (P < 0.05). Expression of FVE and FVE(CS) separately through codon degeneracy substitution (resulting in a reduction in cytosine) led to early flowering (by 8 days and 25 days, respectively). The flowering times of the FVE and FVE(CS) transgenic plants and the Col-0 plants were associated with the number of methylated sites of FVE and FVE(CS), resulting in the differential expression of FVE and FVE(CS). Our findings suggest that the methylation of six key upstream transcription factors of the autonomous pathway in Arabidopsis regulates the expression level of these genes, leading to phenotypic differences in flowering.