Didymosphenia geminata (Didymo) is a benthic freshwater diatom that has been globally expanding its range and extracellular stalk production in freshwater ecosystems. Didymo has been observed in reaches downstream of hypolimnetic reservoir releases in the northeastern US since 2007. This study focused on a newly observed (2013) Didymo occurrence in Pine Creek, a highly forested and unregulated watershed in north-central Pennsylvania. Study objectives included comparing contemporary distribution with historical data to provide insight on historical occurrence, quantifying physicochemical controls on Didymo distribution and benthic mat severity, and examining historical changes in water chemistry that might affect habitat suitability. At present, Didymo cellular distribution is limited to upper reaches of Pine Creek where median soluble reactive phosphorus (SRP) is 2.7 µg/L; median SRP was 4.8 µg/L at sites where Didymo was absent. At the epicenter of distribution in Pine Creek where SRP was consistently <2 µg/L, increased streamflow flashiness and water temperature were associated with decreased benthic mat severity. My results suggest SRP thresholds for Didymo proliferation may vary depending on whether streams are regulated by reservoirs with hypolimnetic releases. Mann–Kendall trends tests of a ∼20-y water chemistry dataset show that orthophosphate and sulfate concentrations decreased while pH increased within Pine Creek, which may have implications for Didymo habitat suitability. Further research is warranted to determine whether improving water quality following the industrial era may facilitate Didymo colonization.