Rice (Oryza sativa L.) is the main food for people in Laos, where it has been grown and eaten since prehistory. Diverse landraces are grown in Laos. ‘Khao Kai Noi’, a landrace favored for its eating quality, is held in the nationwide collection of traditional landraces in the Lao national genebank. Genetic diversity is crucial for sustainable use of genetic resources and conservation. To investigate the genetic diversity of ‘Khao Kai Noi’ for conservation, we genotyped 70 accessions by using 23 polymorphic simple sequence repeat markers. The markers generated 2 to 17 alleles (132 in total), with an average of 5.7 per locus. The total expected heterozygosity over all ‘Khao Kai Noi’ accessions was 0.271. Genetic variation was largest among accessions and smallest within accessions. Khao Kai Noi accessions were classified into three different genetic backgrounds, but there was unclear association between the three inferred population and name subgroups and geographical distribution. Most of the accessions were clustered with temperate japonica and showed genetic relatedness to rice from neighboring provinces of Vietnam, suggesting a Vietnamese origin. The results of this study will contribute to the conservation, core collection and future breeding of the Khao Kai Noi population.