In greenhouse studies of three alloplasmic wheat series, plant height, flowering date and yield per plant were least affected when the native cytoplasm was replaced by donor cytoplasm of the Triticum–Aegilops complex than when replaced by Hordeum chilense cytoplasm. On the other hand, significant differences for seed lutein content were found between euplasmic controls and their respective alloplasmic lines in all the alloplasmic lines studied, underscoring the important role of the cytoplasm to determine the seed carotenoid content. Both T. aestivum subesp. macha and Ae. squarrosa cytoplasms increased the lutein content. They may be the most useful sources of cytoplasmic variability for broadening the genetic diversity of wheat for seed carotenoid content since both cytoplasm types do not produce any detrimental effect on agronomic traits, as previously reported by other researchers. These findings demonstrate the role of the nuclear × cytoplasm interaction in the accumulation of carotenoids in wheat.