Wild rice, Oryza rufipogon, is a genetic resource that can be used to improve cultivated rice, but its populations are now decreasing in terms of both size and number. Extensive research on wild rice has been conducted in Thailand, where two in situ conservation sites have been preserved in natural areas where perennial wild rice predominates. The genetic structure of wild rice populations was investigated by examining both the chloroplast and nucleus genomes at sites of in situ conservation site in Thailand. One accession from an in situ-conserved site was re-sequenced against the chloroplast genome of O. sativa cv. ‘Nipponbare’ to develop chloroplast insertion/deletion (cpINDEL) markers. These cpINDEL markers revealed unique maternal lineages in the in situ-conserved populations upon comparison with other Asian wild rice accessions. Diverse genetic variation was also detected with SSR markers throughout the genome. Three populations differed from each other and also within single populations. The sub-populations within an in situ-conserved population showed a complex population structure due to their multiple maternal lineages and relatively higher number of haplotypes when they maintained a relatively large population size. Such a heterogeneous population would serve as a unique gene pool for rice breeding.