Different basils (Ocimum sp.) and cultivars (28 in 2009 and 32 in 2010) were evaluated for susceptibility to basil downy mildew (Peronospora belbahrii) at the Rutgers Agricultural Research and Extension Center near Bridgeton in southern New Jersey. At the end of each growing season, seed was collected from individual plants and stored for potential downy mildew pathogen detection using real-time polymerase chain reaction (PCR) analysis. Most of the basil cultivars and breeding lines were showing symptoms of basil downy mildew infection at the time of seed collection before the first frost near the end of the production season. Symptoms of basil downy mildew were present on 25 of the 28 (89%) basil lines evaluated in 2009 and 26 of 32 (81%) basil lines tested in 2010 at the time of seed harvest, with sporulation evident on the abaxial surface of infected leaves. Real-time PCR analysis of seed collected from various infected plants detected P. belbahrii on seed of 14 of 25 (56%) basil lines tested in 2009 and 8 of 32 (25%) tested in 2010. Importantly, P. belbahrii was not only detected on seed of sweet basil (Ocimum basilicum) phenotypes but also on seed of ‘Spice’ basil (Ocimum americanum) in 2009 and ‘Sweet Dani Lemon Basil’ basil (Ocimum citriodorum), ‘Holy Red and Green’ basil [Ocimum tenuiflorum (form. sanctum)], ‘Lime’ basil (O. americanum), and again on ‘Spice’ basil in 2010 where no symptoms (i.e., no chlorosis or sporulation) were present on the leaves when seed were collected. This work demonstrates that basil seed, regardless of basil species and whether symptoms are visible on foliage of the basil plant or the plant is immune or resistant to downy mildew, can test positive for the presence of P. belbahrii using a real-time PCR assay following exposure of plants to the pathogen during the natural development of downy mildew under field conditions.