For many organisms inhabiting environments with marked annual variation, seasonal adaptation systems such as diapause are essential for their survival and reproduction. We focused on the two-spotted spider mite, Tetranychus urticae Koch, a model chelicerate species in which diapause has been studied for more than seven decades since photoperiodism was first described by Bondarenko in 1950. In this species, adult females enter reproductive diapause to overwinter when subjected to long-night photoperiods during immature stages. In addition to the reproductive arrest, diapausing females exhibit the distinctive orange body color. However, it remains poorly understood how long-night photoperiods induce these diapause phenotypes at the molecular level. Importantly, intraspecific variation reveals that some populations of T. urticae lack diapause. Here, we applied forward genetic approaches to identify the genes responsible for photoperiodic induction of diapause in T. urticae. We used diapause and non-diapause populations collected in Akita, Japan, and London, Ontario, Canada, respectively. First, we conducted backcrosses between the two populations to investigate the inheritance pattern of diapause. Second, we performed bulked segregant analysis, a linkage-based mapping strategy that identifies candidate genomic regions by comparing allele frequencies between phenotypically contrasting pools. Third, we conducted mRNA sequencing of the two populations to compare transcripts from the candidate genomic region. Finally, we performed RNA interference (RNAi)-mediated functional analysis of a candidate gene identified in the second and third steps, using female deutonymphs during diapause induction. Backcrosses revealed that the non-diapause trait was dominantly inherited and controlled by a single genetic locus. Bulked segregant analysis revealed that one genetic locus on chromosome 1 was strongly associated with diapause. Sixteen genes are located on this locus, and an intergenic region within this locus shows strong homozygosity related to diapause even following multi-generational crossbreeding. RNA-seq reads mapped to the intergenic region indicate transcription of a long non-coding RNA (lncRNA) that showed different transcript lengths between populations. RNAi-mediated functional analysis of the lncRNA showed a 14.4% reduction in the diapause incidence compared to the control. This study contributes to the emerging evidence for lncRNA- mediated regulation of photoperiodic induction of diapause, demonstrating the importance of forward genetic approaches in revealing novel regulatory mechanisms underlying arthropod photoperiodism.