In order to breed a new Pleurotus eryngii cultivar with a large pileus and convex shape, which are favorite traits of customers from Europe and North America, single crosses between monokaryotic mycelia derived from basidiospores of KNR2555 were performed to yield the new cultivar 2×12 chosen by cap shape (convex), diameter of pileus (60.7 mm), and quality (4.9) in a preliminary cultivation. The strain was named Gat_aeryni and was cultivated on a large scale for comparison with Kenneutari 2 ho at the GNARES and at mushroom farms. The yield of Gat_aeryni (71.7 g) was not significantly different from that of Kenneutari 2 ho (71.4 g), and the quality of the new cultivar was 6.8, which was not significantly different from that of a reference cultivar (6.5). Days for harvest, length, and diameter of the pileus in the two cultivars were statistically different by an independent t-test (P < 0.001, P < 0.05, P < 0.001 and P < 0.05 respectively). The new and reference cultivars were discriminated by PCR reactions with a primer set (URP1 and URP10) and simultaneous cultivation.