We examined the conditions for separating and preserving the male and female gametophytes of Kjellmaniella crassifolia. The highest percentage of zygote germination (85%) was on semi-solid medium composed of 1.0% transfer gel agar at $15;^{circ}C$ and $20;{mu}mol/m^2/s$ after a 4-week culture. Zygote germination in PESI liquid medium was 93.5% at $20;^{circ}C$ and $20;{mu}mol/m^2/s$. The maximum zygote growth was $252{pm}19.7;{mu}m$ on 1.0% transfer gel agar at $15;^{circ}C$ and $40;{mu}mol/m^2/s$ after 5-week culture, and was $76.7{pm}2.8;{mu}m$ in PESI liquid medium at $20;^{circ}C$ and $40;{mu}mol/m^2/s$. The respective numbers of separated male and female gametophytes from germinated zygotes were 157 and 93 on 1.0% transfer gel agar and 14 and 28 in PESI liquid medium. The maximum growth of separated male and female gametophytes was $575{pm}28.3;{mu}m$ at $5;^{circ}C$ and $60;{mu}mol/m^2/s$ and $686{pm}35.4;{mu}m$ at $20;^{circ}C$ and $20;{mu}mol/m^2/s$ in PESI liquid medium after 3 weeks, respectively. The highest percentage fertilized was $93.3{pm}5.8%$ at $15;^{circ}C$ and $20;{mu}mol/m^2/s$ in PESI liquid medium. These results show that the best conditions for the separation and preservation of gametophytes (male and female) consisted of culturing on 1.0% transfer gel agar at $15;^{circ}C$ and $20;{mu}mol/m^2/s$.