Diachronic analysis showed no significant changes in the level of genetic diversity occurred over the past 27 years' domestication, which indicated genetic diversity was successfully maintained under on-farm conservation. Rice (Oryza sativa L.) is one of the earliest domesticated crop species. Its genetic diversity has been declining as a result of natural and artificial selection. In this study, we performed the first analysis of the levels and patterns of nucleotide variation in rice genomes under on-farm conservation in Yunnan during a 27-year period of domestication. We performed large-scale sequencing of 600 rice accessions with high diversity, which were collected in 1980 and 2007, using ten unlinked nuclear loci. Diachronic analysis showed no significant changes in the level of genetic diversity occurring over the past 27 years' domestication, which indicated genetic diversity was successfully maintained under on-farm conservation. Population structure revealed that the rice landraces could be grouped into two subpopulations, namely the indica and japonica groups. Interestingly, the alternate distribution of indica and japonica rice landraces could be found in each ecological zone. The results of AMOVA showed that on-farm conservation provides opportunities for continued differentiation and variation of landraces. Therefore, dynamic conservation measures such as on-farm conservation (which is a backup, complementary strategy to ex situ conservation) should be encouraged and enhanced, especially in crop genetic diversity centers. The results of this study offered accurate insights into short-term evolutionary processes and provided a scientific basis for on-farm management practices.