AbstractBecause measuring expansion volume (EV) is simple and inexpensive, popcorn breeders have developed high‐quality single crosses ignoring the contents of zeins, starch, lipids, and cellular wall components in selection. However, some methods of quantification of these quality‐related traits can be applied to popcorn breeding, increasing the selection efficacy for quality. The objectives of this study were to assess methods of zeins and starch quantification that can be used in popcorn breeding, characterize a temperate and a tropical populations for zeins and starch contents and identify candidate genes for these quality‐related traits. We genotyped and phenotyped 286 plants. For quantification of total zeins and zein subunits we choose the ‘lab‐on‐a‐chip’ microfluidic electrophoresis. For quantification of starch and amylose/amylopectin, we choose the Megazyme's Amylose/Amylopectin kit assay. The temperate population has superior EV (36.0%), a higher level of the 19 kDa zein subunit (32.0%), lower levels of the 21, 22 and 27 kDa subunits (−1543.0%, −40.0% and −47.0%, respectively) and no statistical difference for the 10 kDa zein content, relative to the tropical population. Although there are statistical differences between the two populations regarding starch, amylose, and amylose/amylopectin ratio, the differences are not significant (−2.0% to 8.0%). Thirteen candidate genes were identified for the 19 and 22 kDa zeins, two for amylose and one for starch, with emphasis on the genes coding for the 19 and 22 kDa alpha‐zeins, located on chromosome 4. The evaluated quantification methods can be used in popcorn breeding but for a limited number of samples, mainly because costs.