Plant flowering time is affected by endogenous and exogenous factors, but its polymorphism among different populations of a species has not been fully explained yet. 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 showed that polymorphism in the flowering time among the Arabidopsis populations ranged from 19 to 55 days was significantly correlated with methylation of the coding regions of six upstream genes in the autonomous pathway, FVE, FY, FLD, PEP, HDA5 and PPR 39-1 and their relative expression levels (P < 0.05). Expression of selected FVE and FVE(CS) separately through codon degeneracy substitution of cytosine led to earlier flowering of transgenic plants by 8 days and 25 days, respectively. An accurate determination of methylated sites of FVE and FVE(CS) among those transgenic plants and recipient Col-0 verified the close relationship between their methylation number, expression and the flowering times. Our findings firstly suggest that the methylation variation of those six key upstream transcription factors regulates the gene expression level of the autonomous pathway, leading to phenotypic differences in flowering time of Arabidopsis. The FVE(CS) and FVE genes in transgenic plants tend to be hypermethylated, and this could be a protective mechanism for plants. However, modification of gene sequence through codon degeneracy substitution to reduce cytosine can avoid hypermethylated transferred-genes in transgenic plants. It may be possible to partially regulate the flowering of plants by modified trans-epigenetic technology.