We characterized deep chlorophyll layers (DCLs), previously unknown, in Lake of the Woods (Canada), a complex hydrological system with strong trophic, physical, and chemical gradients. Of 42 sites, five (12 %) contained at least one dense, thin metalimnetic peak – often more, overlapping or vertically stratified. In spring, highest biomass (>4 mg/L) was found at 3 m in Bigstone Bay, composed of a mixed phytoplankton community. Other, deeper, spring DCLs were dominated by the diatom Cyclotella (∼1 mg/L, 21 m), dinoflagellate Gymnodinium (>1 mg/L, 17 m), cyanobacterium Dolichospermum (>3 mg/L, 10 m), or cyanobacterium Woronichinia (>1 mg/L, ∼8 m). In summer, the highest biomass of a DCL was in Yellow Girl Bay (>21 mg/L), over five times higher than spring and higher than large surface blooms from the shallow, eutrophic south (Sabaskong Bay ∼19 mg/L). Summer DCLs (∼7 m) were not as deep as spring, owing to reduced light penetration (2.5-fold lower Zeu:Zmix). Most summer DCLs were dominated by Dolichospermum (max. >16 mg/L) but other species were also high (∼1 mg/L biomass), including cyanobacterium Aphanizomenon, diatom Aulacoseira, and cryptophyte Cryptomonas. Euphotic zone in these embayments intersected with a nutrient-enriched (PO₄, Fe, Mn, Si) hypoxic hypolimnion, conditions conducive to DCLs. Other drivers of composition and activity of DCLs remain to be elucidated. These high levels of DCL biomass, up to fivefold higher than near-surface, have routinely gone unreported. Their inclusion in establishing baselines and tracking change would inform lake management decisions and qualify expectations for ecosystem response.