Ecological speciation has long been noted as a central topic in the field of evolutionary biology, and investigation into the relative importance of ecological and geographical factors is becoming increasingly emphasized. We surveyed genetic variation of 277 samples from 25 populations of nine Rhododendron species within Tsutsusi subgenus in Taiwan using simple sequence repeats of expressed sequence tags. Bayesian clustering revealed four genetic lineages: (1) the Rhododendron simsii, Rhododendron kanehirai, and Rhododendron nakaharae lineage (lineage 1); (2) the R h o d o d e n d ro n l o n g i p e r u l a t u m , R h o d o d e n d ro n breviperulatum, and Rhododendron noriakianum lineage (lineage 2); (3) the Rhododendron rubropilosum lineage (lineage 3); and (4) the Rhododendron oldhamii lineage (lineage 4).Asymmetric introgressions were found from lineage 3 into lineages 1 and 2 (introgressed lineages). Genetic admixture of non-R. oldhamii species was also revealed by a neighborjoining tree. Variation partitioning showed that environment explained much larger portions of genetic variation than geography between non-introgressed lineages (i.e., between R. oldhamii and other lineages). However, the Mantel and partial Mantel tests and the multiple matrix regression with randomization found that isolation-by-distance played a more important role than isolation-by-environment (IBE) in contributing to genetic variation in most between lineage comparisons. Nevertheless, strong IBE was found when compared between non-introgressed lineages of R. oldhamii and R. rubropilosum, suggesting post-speciation ecological divergence. Several environmental variables, including annual mean temperature, aspect, isothermality, seasonal precipitation, slope, and soil pH, could be important ecological drivers involved in reproductive isolation between R. oldhamii and non-R. oldhamii species within the Tsutsusi subgenus.