Knowledge about genetic diversity among Brassica napus cultivars developed for many growing regions and their possible use as potential inbred lines for hybrid seed production is limited. We studied the genetic diversity and relationships among B. napus accessions using Sequence Related Amplified Polymorphism (SRAP) markers, which preferentially amplify open reading frames. A total of 60 spring-type B. napus accessions were screened using 20 SRAP primers, which revealed 162 polymorphic fragments with an average of eight markers per primer combination. Genetic similarity estimates ranged from 40 to 100, which indicated sufficient diversity among the accessions. The majority of the accessions were uniquely identified by the markers with the exception of near-isogenic inbred lines. Cluster analysis displayed five major groups. The first major cluster comprised 23 accessions mostly of Australian origin, whereas the second cluster included 13 accessions mostly of Canadian origin. The accessions in the first and second clusters were identified as maintainers of cytoplasmic male sterility. The two restorer lines R-111 and R-101 along with their corresponding backcross progeny constituted the third cluster. Scandinavian cultivars made the fourth separate cluster. One cultivar Salam and its respective inbred line were the most divergent lines. Variations in the number of markers between open-pollinated cultivars and their respective selfed inbred lines were also observed. The clustering pattern mostly supported their respective pedigree and characteristic traits. Genetic diversity in genetically distinct groups in the tested maintainer and restorer lines can be exploited for hybrid development in B. napus.