Over the past century, industrialization and urban practices have resulted in the contamination of the Great Lakes ecosystem—the world's largest surface freshwater system—that provides drinking water and recreation to more than 40 million residents. In 2010, the Great Lakes Restoration Initiative was launched to accelerate efforts to protect and restore the Great Lakes and surrounding areas. Funded by GLRI, the Agency for Toxic Substances and Disease Registry initiated the Biomonitoring of Great Lakes Populations (BGLP) program. The objective of the program is to assess human exposure to legacy and emerging contaminants in the Great Lakes by measuring the body burden of contaminants in potentially susceptible populations. The BGLP program consists of a series of cross-sectional studies carried out collaboratively with states that are funded through ATSDR. The first BGLP Program (BGLP-I) began in 2010 and was completed in September 2015 through cooperative agreements with state health departments in Michigan, Minnesota, and New York. The three state programs targeted susceptible adult populations living in designated areas of contamination. Contaminants measured in all populations include mercury, lead, mirex, hexachlorobenzene, dichlorodiphenyltrichloroethane, and selected polychlorinated biphenyl congeners. In addition, some chemicals of emerging concern, such as per- and polyfluoroalkyl substances, were measured in several populations. The biomonitoring results helped guide public health actions to mitigate chemical exposures in these vulnerable Great Lakes populations. We provide an overview of the BGLP-I program's study populations, designs, and general methods. This overview provides a lead-in for subsequent manuscripts that present human biomonitoring data for legacy and emerging contaminants in culturally diverse susceptible populations—i.e., shoreline anglers, sport anglers, American Indians, and Burmese immigrants—residing in seven areas of concern.