Starch is a product of photosynthetic activities in leaves. Wheat yields largely depend on photosynthetic carbon fixation and carbohydrate metabolism in flag leaves. The mapping of quantitative trait loci (QTLs) associated with flag leaf starch content (FLSC) in wheat (Triticum aestivum L.) was completed using unconditional and conditional QTL analyses. The FLSC of this population during the early grain-filling stage was measured at six stages in six environments. Combining unconditional and conditional QTL mapping methods, eight unconditional QTLs and nine conditional QTLs were detected, with five QTLs identified as unconditional and conditional QTLs. Four unconditional QTLs (i.e. qFLS-1B, qFLS-1D-1, qFLS-4A, and qFLS-7D-1) and one conditional QTL (i.e. qFLS-3A-1) were identified in two of six environments. Two QTLs (qFLS-1D-2 and qFLS-7D-1), which significantly affected the FLSC, were identified using the unconditional QTL mapping method, while three QTLs (i.e. qFLS-1A, qFLS-3A-1, and qFLS-7D-1) were detected using the conditional QTL mapping method. Our findings provide new insights into the genetic mechanism and regulatory network underlying the diurnal FLSC in wheat.