Two homoeologous QTLs for number of spikelets per spike (SPS) were mapped on chromosomes 7AL and 7BL using two wheat MAGIC populations. Sets of lines contrasting for the QTL on 7AL were developed which allowed for the validation and fine mapping of the 7AL QTL and for the identification of a previously described candidate gene, WHEAT ORTHOLOG OF APO1 (WAPO1). Using transgenic overexpression in both a low and a high SPS line, we provide a functional validation for the role of this gene in determining SPS also in hexaploid wheat. We show that the expression levels of this gene positively correlate with SPS in multiple MAGIC founder lines under field conditions as well as in transgenic lines grown in the greenhouse. This work highlights the potential use of WAPO1 in hexaploid wheat for further yield increases. The impact of WAPO1 and SPS on yield depends on other genetic and environmental factors, hence, will require a finely balanced expression level to avoid the development of detrimental pleiotropic phenotypes.