The Northern Highlands Lake District (NHLD) is one of the few regions in the world with periodic comprehensive water chemistry data from hundreds of lakes spanning almost a century. Birge and Juday directed the first comprehensive assessment of water chemistry in the NHLD, sampling more than 600 lakes in the 1920s and 30s. These surveys have been repeated by various agencies and we now have data from the 1920s (UW), 1960s (WDNR), 1970s (EPA), 1980s (EPA), 1990s (EPA), and 2000s (NTL). The 28 lakes sampled as part of the Regional Lake Survey have been sampled by at least four of these regional surveys including the 1920s Birge and Juday sampling efforts. These 28 lakes were selected to represent a gradient of landscape position and shoreline development, both of which are important factors influencing social and ecological dynamics of lakes in the NHLD. This long-term regional dataset will lead to a greater understanding of whether and how large-scale drivers such as climate change and variability, lakeshore residential development, introductions of invasive species, or forest management have altered regional water chemistry. Zooplankton samples were taken at approximately the deepest part of each lake, via a vertical tow with a Wisconsin net. Count of individuals and presence absence data for all lakes in the study region are provided here.