Endoreduplication is the process where a cell replicates its genome without mitosis and cytokinesis, often followed by cell differentiation. This alternative cell cycle results in various levels of endoploidy, reaching 4× or higher one haploid set of chromosomes. Endoreduplication is found in animals and is widespread in plants, where it plays a major role in cellular differentiation and plant development. Here, we show that variation in endoreduplication between Arabidopsis thaliana accessions Columbia-0 and Kashmir is controlled by two major quantitative trait loci, ENDO-1 and ENDO-2. A local candidate gene association analysis in a set of 87 accessions, combined with expression analysis, identified CYCD5;1 as the most likely candidate gene underlying ENDO-2, operating as a rate-determining factor of endoreduplication. In accordance, both the overexpression and silencing of CYCD5;1 were effective in changing DNA ploidy levels, confirming CYCD5;1 to be a previously undescribed quantitative trait gene underlying endoreduplication in Arabidopsis.