Yellow-phase American eel (Anguilla rostrata) upstream migration is temporally punctuated, yet migration chronology within diel time periods is not well-understood. This study examined diel periodicity, chronology, and total length (TL) of six multi-day, high-count (285–1,868 eels) passage events of upstream migrant yellow-phase American eels at the Millville Dam eel ladder, lower Shenandoah River, West Virginia during 2011–2014. We categorized passage by diel periods (vespertine, nocturnal, matutinal, diurnal) and season (spring, summer, late summer/early fall, fall). We depicted passage counts as time-series histograms and used time-series spectral analysis (Fast Fourier Transformation) to identify cyclical patterns and diel periodicity of upstream migration. We created histograms to examine movement patterns within diel periods for each passage event and fit normal mixture models (2–9 mixtures) to describe multiple peaks of passage counts. Periodicity of movements for each passage event followed a 24-h activity cycle with mostly nocturnal movement. Multimodal models were supported by the data; most modes represented nocturnal movements, but modes at or near the transition between twilight and night were also common. We used mixed-model methodology to examine relationships among TL, diel period, and season. An additive-effects model of diel period + season was the best approximating model. A decreasing trend of mean TL occurred across diel movement periods, with the highest mean TL occurring during fall relative to similar mean values of TL for spring, summer, and late summer/early fall. This study increased our understanding of yellow-phase American eels by demonstrating the non-random nature of their upstream migration.