twilight is a Bioconductor compatible package for analysing the statistical significance of differentially expressed genes. It is based on the concept of the local false discovery rate (FDR), a generalization of the frequently used global FDR. twilight implements the heuristic search algorithm for estimating the local FDR introduced in our earlier work. In addition to the raw significance measures, it produces diagnostic plots, which provide insight into the extent of differential expression across genes.