The tropical butterfly Bicyclus anynana shows wide variation in the size of the eyespots on the ventral side of its wings. Differences in the environmental temperature during late larval and early pupal stages are a major source of this variation, but variation also exists within temperatures. Using lines selected at a single temperature for large and small eyespots, and a number of crosses derived from these lines, we studied the genetic basis of eyespot size in B. anynana. We applied Lande's modification of the Castle-Wright (C-W) estimator to estimate the minimum number of genes contributing to the difference between the two lines. Estimates indicated that at least five genes are involved. As the C-W estimator is based on a number of simplifying assumptions, we tested to what extent one of these assumptions (additive gene action) is actually met. Joint-scaling tests indicated that the assumption of additivity is not satisfied and that dominance and probably epistasis play a role. Because reciprocal crosses were available we looked for evidence for sex-linkage and cytoplasmic effects. No evidence for cytoplasmic effects was found, but the data were consistent with the presence of one or more loci on the X-chromosome. The results are discussed in the context of the current model of eyespot formation.