AbstractEvolution can be contingent on history, but we do not yet have a clear understanding of the processes and dynamics that govern contingency. Here, we performed the second phase of a two-phase evolution experiment to investigate features of contingency. The first phase of the experiment was based onEscherichia coliclones that had evolved at the stressful temperature of 42.2 °C. The Phase 1 lines generally evolved through two adaptive pathways: mutations ofrpoB, which encodes the beta subunit of RNA polymerase, or throughrho, a transcriptional terminator. We hypothesized that epistatic interactions within the two pathways constrained their future adaptative potential, thus affecting patterns of historical contingency. Using ten differentE. coliFounders representing both adaptive pathways, we performed a second phase of evolution at 19.0 °C to investigate how prior genetic divergence or adaptive pathway (rpoBvs.rho) affects evolutionary outcomes. We found that phenotype, as measured by relative fitness, was contingent on founder genotypes and pathways. This finding extended to genotypes, becauseE. colifrom different Phase 1 histories evolved by adaptive mutations in distinct sets of genes. Our results suggest that evolution depends critically on genetic history, likely due to idiosyncratic epistatic interactions within and between evolutionary modules.