The opossum shrimp, Mysis diluviana, is an important member of the offshore food webs of the Laurentian Great Lakes, but its response to ecosystem changes that have occurred over the past several decades is not well understood. We combined the data of four long-term sampling programs, adding several years of data (post and prior) to previously published analyses to offer a longer-term, cross-basin analysis of M. diluviana populations in the Great Lakes from 1997 to 2019. Densities were high in lakes Superior and Ontario (summer values 100–300/m²), high and variable but declining (from 200–300/m² in 1997–2004 to less than 100/m² in 2017–2019) in Lake Michigan, low (∼20–50/m² since 2005) in Lake Huron, and very low in shallower eastern Lake Erie (<1/m²). Biomass showed similar trends. Life history parameters (mortality, fecundity, and growth) were consistently highest in eastern Lake Erie, followed by lakes Ontario, Michigan, Huron, and Superior. Generation time was 1 year in Lake Erie and 2 years in the other lakes. Cross-basin relationships between annual M. diluviana areal densities and food indices (chlorophyll-a concentration and zooplankton biomass) were non-linear, increasing with food levels up to about 250 mysids/m² and about 650 mg dry wt/m². Annual growth rates were also positively correlated to both food indices in the four deep lakes, but fecundity and mortality rates were not. Our results suggest food availability is a primary factor predicting M. diluviana density and biomass. Density-dependent mortality and fish predation could explain some of the inter-lake differences, but these relationships could benefit from further investigations.