Inheritance of pollen colour was studied in sunflower (Helianthus annuus L.) using three distinct pollen colour morphs: orange, yellow and white-cream. Orange is the most common colour of sunflower pollen, while the yellow morph is less frequent. These two types were observed in the inbred lines F11 and EF2L, respectively. White-cream pollen is a rare phenotype in nature, and was identified in a mutant, named white-cream pollen, recovered in the R2 generation of an in vitro regenerated plant. The F11 inbred line was used as starting material for in vitro regeneration. The carotenoid content of these three pollen morphs differed, and was extremely reduced in white-cream pollen. The phenotype of F1 populations obtained by reciprocal crosses revealed that the orange trait was dominant over both white-cream and yellow. Segregation of F2 populations of both crosses, orange × yellow and orange × white-cream, approached a 3:1 ratio, indicating the possibility of simple genetic control. By contrast, a complementation cross between the two lines with white-cream and yellow pollen produced F1 plants with orange pollen. The F2 populations of this cross-segregated as nine orange: four white-cream: four yellow. A model conforming to the involvement of two unlinked genes, here designated Y and O, can explain these results. Accessions with yellow pollen would have the genotype YYoo, the white-cream pollen mutant would have yyOO and the accession with orange pollen would have YYOO. Within F2 populations of the cross white-cream × yellow a new genotype, yyoo, with white-cream pollen was scored. The results of the cross yyoo × YYoo produced only F1 plants with yellow pollen, supporting a recessive epistatic model of inheritance between two loci. In this model, yy is epistatic on O and o. In F2 populations, the distributions of phenotypic classes suggested that the genetic control of carotenoid content is governed by major genes, with large effects segregating in a background of polygenic variation. These three pollen morphs can provide insight into the sequence in which genes act, as well into the biochemical pathway controlling carotenoid biosynthesis in anthers and the transfer of these different pigments into pollenkitt.