The culm (particularly the flag leaf) and the ear are believed to play a major role in providing assimilates for grain filling in wheat. However, the results obtained in the past varied depending on the methodology applied. Three different methodologies were compared that aimed to assess the relative contribution of the culm (photosynthetic organs below the ear) and the ear to grain filling. The first two consisted of applications of photosynthesis inhibition treatments, including the use of the herbicide DCMU and organ shading. The third was a non-intrusive method that compared the carbon isotope composition (δ13C) of mature kernels with the δ13C of the water-soluble fraction of the peduncle, awns and glumes. Several advanced CIMMYT lines were tested under good agronomic conditions. The δ13C approach assigned a higher photosynthetic contribution to the ear than to the culm. However, some methodological considerations should be taken into account when applying the δ13C approach, particularly the sampling method used, in order to prevent post-harvest respiration. The shading approach assigned a similar contribution to the ear as to the culm. The DCMU approach assigned a greater role to the culm but herbicide application to the culm affected the ear, thus biasing the final grain weight. Moreover DCMU and shading approaches may cause compensatory effects which overestimated the contribution of unaffected organs. This study may help to develop precise phenotyping tools to identify physiological traits such as ear photosynthesis that could contribute towards increasing grain yield.