It has been hypothesized that efficient exclusion of methylated retrotransposons and repeated DNA region is one of the rapid and cost-effective approaches for comprehensive gene discovery in large genome size of maize. Three kinds of methylation-sensitive restriction enzymes, HapII, MspI and McrBC, were used to identify the restriction frequency of cytosine methylation sites in maize genome. Roughly 60% of total maize genomic DNA was restricted less than 500bp by McrBC, and the most of restricted small size fraction was composed retrotransposon. In order to validate the efficient construction of gene-rich shotgun library, we compare two gene-rich methyl-filtration shotgun libraries using in vivo and in vitro methyl-filtration system. The size selected DNA fraction by Sau3A-McrBC enzyme treated was very stable and has not appeared modification in E. coli, but most insert DNA size of partially digested with Sau3A were decrease less than 500bp by bacterial methylation-modification system. In compare of retroelements portion, A 44.6% of the sequences were retroelement in unmethyl-filtered library, and the most of them was Copia type, such as Prem, Opie and Ji. The portion of retroelement was drastically decreased to 25% and 20% by in vivo and in vitro filtration system, respectively.