In this study, monokaryons of “Heukari” (Pleurotus ostreatus) and “Hosan” (Pleurotus pulmonarius) were separated to remove the cell wall, and a cross-species protoplast fusion was developed through chemical treatment with polyethylene glycol . The protoplast-fused PF160306 and PF160313 strains have a culture period of 10 and 2 days shorter than that of the “Heuktari” and “Hosan” cultivars, respectively. Furthermore, the growth of the strains was faster than that of the existing cultivars. The yield was 135.9 g per bottle, which was approximately 8% higher than that of the commercially available “Hosan” cultivar; however, it was not statistically significant. A growth survey was conducted after treatment at five temperatures (15, 18, 21, 23, and 25oC). The growth of the strains accelerated with the increase in temperature. However, at 21oC, the yellow color of pileus was the brightest. Band pattern, assessed using URP Primer 7, was similar to that of the “Hosan” cultivar. The DPPH radical scavenging capacity and polyphenol content were 62.5% and 43.5 mg/mL, respectively, for “Sunjung” and 65.7% and 49.9 mg/mL, respectively, for PF160313. Furthermore, the antihypertensive activities of the “Sunjung” cultivar and PF160313 were similarly high at 74% and 75%, respectively. In conclusion, cross-species hybridization via the protoplast fusion technique can be used for obtaining primary data for mushroom breeding to develop new varieties. In addition, the protoplast fusion technique might aid in expanding the market for yellow mushrooms.