Orestias ascotanensis is a killifish endemic to the Ascotán salt pan in the Chilean Altiplano, where it inhabits 12 springs with different degrees of isolation. This species is a suitable model for studying the effect of serial geographical isolations on the differentiation process among populations. The present study examines the genetic variation and structure of the species using mitochondrial DNA control region sequences and eight microsatellite loci, analyzing populations across its distribution range. The evaluation of genetic variation revealed high levels of diversity within the species. The genetic structure analysis showed the existence of four differentiated groups: two groups were formed by the springs located in the northern and southern extremes of the salt pan and two groups were found in the centre of the salt pan. The latter two groups were formed by several springs, most likely as a consequence of the South American summer monsoon that could connect them and allow gene flow. The patterns of genetic differentiation appear to be determined based on the physical isolation of the populations. This isolation may be the result of a combination of factors, including geographical distance, a historical decrease in water levels and altitude differences in the springs of the salt pan. Therefore, this system is a rare example in which hydrological factors can explain genetic differentiation on a very small scale.