Heritable variation in gene expression is common within and between species. This variation arises from mutations that alter the form or function of molecular gene regulatory networks that are then filtered by natural selection. High-throughput methods for introducing mutations and characterizing their cis- and trans-regulatory effects on gene expression (particularly, transcription) are revealing how different molecular mechanisms generate regulatory variation, and studies comparing these mutational effects with variation seen in the wild are teasing apart the role of neutral and non-neutral evolutionary processes. This integration of molecular and evolutionary biology allows us to understand how the variation in gene expression we see today came to be and to predict how it is most likely to evolve in the future.