Flowering time has biological and agricultural significance for crops. In Upland cotton (Gossypium hirsutum L.), photoperiodic sensitivity is a major obstacle in the utilization of primitive accessions in breeding programs. Quantitative trait loci (QTLs) analysis was conducted in two F2 populations from the crosses between a day-neutral cultivar Deltapine 61 (DPL61) and two photoperiod sensitive G. hirsutum accessions (T1107 and T1354). Node of first fruiting branch (NFB) was used to measure relative time of flowering. Different flowering time genetic patterns were observed in the two populations. Two QTLs were found across five scoring dates, accounting 28.5 (qNFB-c21-1) and 15.9% (qNFB-c25-1) of the phenotypic variation at the last scoring date in Pop. 1107 (DPL61 by T1107); whereas, one major QTL (qNFB-c25-1) can be detected across five scoring dates, explained 63.5% of the phenotypic variation at the last scoring date in Pop. 1354 (DPL61 by T1354). QTLs with minor effects appeared at various scoring date(s), indicating their roles in regulating flowering at a lower or higher node number. Genetic segregation analysis and QTL mapping results provide further information on the mechanisms of cotton photoperiodic sensitivity.