The underlying mechanisms for hybrid vigor or heterosis are elusive. Here we report a population of recombinant inbred lines (RILs), derived from the two ecotypes, Col and Ler, which can serve as a permanent resource for studying the molecular basis of hybrid vigor in Arabidopsis. Using a North Carolina mating design III (NCIII), we determined the additive and dominant nature of gene action in this population. We detected heterosis among crosses of RILs with one of the two parents (Col and Ler) and analyzed genotypes and heterozygosities for RILs and test cross families (RILs crossed to Col and Ler) using a total of 446 published molecular markers. The performance of RILs and additive and dominant components in the test cross families were used to analyze QTLs for 16 traits, using QTL cartographer and composite interval mapping with 1,000 permutations for each trait. Our data suggest that locus-specific and/or genome-wide differential heterozygosity, including epistasis, plays an important role in the generation of the observed heterosis. Furthermore, the hybrid vigor occurred between two closely related ecotypes, and provides a general mechanism for novel variation generated between genetically similar materials.