We collected 32 maize inbred lines from eastern cereal and oilseed research center in Canada to develop new maize varieties. We also evaluated genetic diversity, genetic relationships, and population structure using 35 SSR markers. A total of 269 alleles were revealed in 35 loci with an average of 7.69 and a range between 3 and 15 alleles per locus. The genetic diversity values varied from 0.176 to 0.889 with an average of 0.691. The polymorphic information content varied from 0.171 to 0.879 with an average of 0.659. Population structure analysis indicated that 32 Canadian maize inbred lines comprised four major groups and one admixed group based on a membership probability threshold of 0.80. The four major groups contained 13, 2, 5 and 2 maize inbred lines, respectively. From genetic relationships analysis, the all inbred lines were divided into three main groups at 26% genetic similarity. Group I included 22 inbred lines, and Group II included 9 inbred lines. Group III consist of only one inbred line. The results in this study would be useful for the improvement and development of new cultivars, planning crosses for hybrids or development of inbred line in maize breeding program