Molecular characterization information on genetic diversity, population structure and genetic relationships provided by this research will help maize breeders to better understand how to utilize the current CML collection. CIMMYT maize inbred lines (CMLs) have been widely used all over the world and have contributed greatly to both tropical and temperate maize improvement. Genetic diversity and population structure of the current CML collection and of six temperate inbred lines were assessed and relationships among all lines were determined with genotyping-by-sequencing SNPs. Results indicated that: (1) wider genetic distance and low kinship coefficients among most pairs of lines reflected the uniqueness of most lines in the current CML collection; (2) the population structure and genetic divergence between the Temperate subgroup and Tropical subgroups were clear; three major environmental adaptation groups (Lowland Tropical, Subtropical/Mid-altitude and Highland Tropical subgroups) were clearly present in the current CML collection; (3) the genetic diversity of the three Tropical subgroups was similar and greater than that of the Temperate subgroup; the average genetic distance between the Temperate and Tropical subgroups was greater than among Tropical subgroups; and (4) heterotic patterns in each environmental adaptation group estimated using GBS SNPs were only partially consistent with patterns estimated based on combining ability tests and pedigree information. Combining current heterotic information based on combining ability tests and the genetic relationships inferred from molecular marker analyses may be the best strategy to define heterotic groups for future tropical maize improvement. Information resulting from this research will help breeders to better understand how to utilize all the CMLs to select parental lines, replace testers, assign heterotic groups and create a core set of breeding germplasm.