A good working knowledge of seed germination requirements is important for plant establishment for restoration and experimentation, particularly in wild plant species. Some seeds require a period of after-ripening and cold stratification before germination will occur. In this study we examined the effects of after-ripening and stratification time on seeds from three disjunct populations of Arabidopsis lyrata ssp. lyrata, from serpentine and limestone sand substrates of the Mid-Atlantic region of the United States. Differences among populations were evaluated through seed germination percentage and rate. Overall germination percentage and rate significantly varied by population, with seeds from the two serpentine populations having a lower germination percentage and rate than seeds from the limestone sand population. After-ripening time also significantly affected germination percentage and rate, with both measurements generally being the highest in 4 and 8 month-old seeds. Stratification did not generally alter germination percentage compared to the untreated control. However, stratification appeared to have the greatest effect on the germination rate of seeds that had after-ripened for 4 months, but the number of days of stratification required was not consistent across populations or across years. The variation observed among the populations of A. lyrata ssp. lyrata tested is a starting point for understanding how these differences developed and how they contribute to the success of each population in its native environment.