Changes in gene expression are an important source of phenotypic differences within and between species. Differences in RNA abundance can be readily quantified between genotypes using a variety of tools, including microarrays, quantitative real-time PCR, cDNA sequencing, and in situ hybridization, but determining the genetic basis of heritable expression differences has historically been less straightforward. Genetic changes that affect RNA abundance can be broadly classified into two groups depending on how they affect gene expression: cis-acting changes affect expression of a single allele in a diploid cell and are typically located close to the affected gene in the genome, whereas trans-acting changes affect expression of both alleles of a gene in a diploid cell and can be located virtually anywhere within a genome. By comparing relative expression of two alleles in an F(1) hybrid with relative expression between the two parental genotypes, the net effects of cis- and trans-acting changes can be discerned. Here, I describe how pyrosequencing can be used to obtain relative gene-specific and allele-specific expression. I also describe how such data can be used to infer the relative contribution of cis- and trans-acting changes to expression differences between genotypes.