A high level of genetic purity in crop varieties must be achieved and maintained for agronomic performance, encouraging investment and innovation in plant breeding and ensuring that the improvements in productivity and quality imparted by breeders are delivered to the consumer. Since the success of hybrid seed production is dependent upon the genetic purity of the parental lines, in this study, the experimental F1expmaize hybrid and its parental inbreeds were used as a model system to examine the discriminative power of morphological, biochemical and SSR markers for seed purity assay. The highest number of off-type plants was estimated by morphological markers. According to the comparison of prolamins and albumins banding patterns of parental and derived F1expseeds, genetic impurities could not be detected. Molecular analysis detected two types of genetic profile irregularity. Beside its use for verifying varieties of maize, report onumc1545 primer pair ability to detect non-specific bands (i.e., off-types), in both the maternal component and F1exp, which is the first report on this issue yet, strongly supports the recommendation of this SSR marker use for more accurate and time-efficient maize hybrids and parental lines genetic pyrity testing.