Differences in diapause traits can result in the seasonal reproductive isolation of host plant-associated insect populations and thereby facilitate the population divergence. The striped stem borer, Chilo suppressalis, has two host plant-associated populations: rice population and water-oat population. Several studies have found evidence that seasonal reproductive isolation between these populations is at least partially due to interpopulation differences in diapause. However, there still lack unambiguous evidence comparing characteristics of diapause induction for both populations. We compared the photoperiodic response and the age of peak photoperiod sensitivity of these populations and used RNA-Seq to compare the molecular response of diapause induction between populations. The photoperiodic response of the two populations differed at 25°C; the critical night length of larvae from the rice population was 11h and 20min, whereas no obvious critical night length was in those from the water-oat population. In rice population, larvae were most sensitive to photoperiod at 9-12days of age, whereas in water-oat population, larvae were the most sensitive to photoperiod at 9-10days of age. The RNA-Seq results indicated that there were several differences in the molecular response of diapause induction and small overlap in differentially expressed genes (DEGs) between populations. Furthermore, GO analysis indicated that both rice and water-oat population's DEGs were significantly enriched in heme and iron binding. Besides, water-oat population's DEGs were significantly enriched in metabolizing nutrients but rice population's DEGs do not. Thus, our results described differences in diapause induction between rice and water-oat populations of C. suppressalis which could affect the timing of diapause and thereby contribute to the seasonal reproductive isolation of these host plant-associated populations. In conclusion, this work suggests that difference in diapause induction could promote the population divergence in insects associated with different host plants.