We compared two haploid genotypes of one Ciona savignyi individual and identified codons at which these genotypes differ by two nonsynonymous substitutions. Using the C. intestinalis genome as an outgroup, we showed that both substitutions tend to occur in the same genotype. Only in 53 (34.4%) of 154 codons, one substitution occurred in each of the two genotypes, although 77 (50%) of such codons are to be expected if substitutions were independent. We considered two feasible evolutionary causes for the observed pattern: substitutions driven by positive selection and compensatory substitutions, as well as several potential biases. However, none of these explanations is fully compelling, and data on multiple genotypes of C. savignyi would help to elucidate the causes of this pattern.