Following the transition from outdoor seedbeds to indoor greenhouse systems in the 1980s, flue-cured tobacco seedlings have been overwhelmingly produced in expanded polystyrene (EPS) trays. In 2015, hard plastic float trays were marketed in the United States. Research was conducted in North Carolina to compare the performance of three 288 cell tray types: hard plastic, new EPS, and five-year-old EPS. In addition, four soilless media brands (Carolina Gold, Carolina Choice, Premier Pro BF, and Sunshine LT5) were evaluated within each tray type. Treatments were imposed in a single tobacco greenhouse in a randomized complete block design with a factorial treatment arrangement replicated six times. Following tray fill, percent dry cell, percent seedling emergence, and percent spiral root were evaluated from one to 18 days after seeding (DAS), depending upon parameter. Fifty days after seeding, percent total plants, percent usable plants, stem diameter, and stem height were quantified. Results indicate that new EPS trays were more likely to contain a greater number of dry cells 9 DAS, specifically when filled with Carolina Gold (9%) or Sunshine LT5 (14%) media. Reduced seedling emergence was also observed in the same treatments 13 DAS. No differences were observed in percent spiral root among tray types 18 DAS; however, spiral root was lowest in the Sunshine LT5 media (0.61%), most likely due to poor seed emergence from dry cells. Fifty DAS, older EPS trays were found to produce more total and usable plants than new EPS or hard plastic trays. In addition, the Carolina Gold, Carolina Choice, and Premier Pro BF soilless media brands produced a greater number of total and usable seedlings exhibiting preferred quality. Preliminary results indicate that there are numerous tray and soilless media combinations suitable for tobacco transplant production.