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<i>Stilpnolepis centiflora</i> is an endemic annual herb in the Asteraceae family found across five sand deserts in Northwest China. We aimed to investigate the genetic structure of <i>S. centiflora</i> and attempt to link species evolution with desert formation during the Pleistocene era. We used sequence data from nuclear and chloroplast genes to investigate genetic diversity among 28 populations. We analyzed sequence data using network analysis, spatial analysis of molecular variance (SAMOVA), and a Mantel test. We then used a molecular clock to place the genetic patterns in a temporal framework and tested for signals of expansion using neutrality tests and by determining mismatch distributions. Six distinct haplotypes and 31 ribotypes were identified. Significant chloroplast DNA population subdivision was detected (G_ST = 0.952; N_ST = 0.976), but only moderate nrDNA subdivision (G_ST = 0.360; N_ST = 0.579) was detected. SAMOVA revealed four diverging groups of related haplotypes, coinciding with the boundaries of deserts. Molecular dating suggests that the clades representing different deserts diverged from 1.2 to 0.20 Ma, concordant with the Kun-Huang Movement of Qinghai Tibet Plateau uplift and a glacial event (Naynayxungla) during the Middle–Late Pleistocene. The disjunction of <i>S. centiflora</i> among different deserts was correspondingly reflected in the examined genetic traits with consistent spatiotemporal evolution between species and deserts. Therefore, the evolutionary dynamics of <i>S. centiflora</i> appear to have been driven by geological movement and climate change. The patterns described here are potentially useful to conservation biologists and may serve as a model for other sand-obligate organisms found in the deserts of Northwest China.