In this study, we made a population with high biological efficiency (BE) to investigate the complex genetic architecture of yield-related traits in Agaricus bisporus. MB-013 crossed between bisp 015-p2 and bisp 034-p2, had high BE. Additionally MB-013 was an intervarietal hybrid that intercrosses with A. bisporus var. burnettii, bisp 015, and A. bisporus var. bisporus, bisp 034. One hundred and seventy homokaryons were selected using the cleaved amplified polymorphic sequence (CAPS) markers (PIN primer/HaeIII) from 300 single spore isolates (SSIs). One hundred BC₁F₁ hybrids were obtained by crossing the homokaryons of MB-013 with bisp15-p1. The population of 100 BC1F1 hybrids is suitable for analyses of BE.