Determination of appropriate stages of flower emasculation and flower development when self-pollination occurs in tomato (Solanum lycopersicum L.) flowers is important to increase the degree of success of cross pollination. The experiment was conducted from July 2015 to June 2017. Seven tomato varieties (as female parents) and 1 genotype (as pollen source) were evaluated. Stages of flower emasculation (3 levels) were: 1) emasculation of immature flowers (light yellow petal) (Stage I); 2) emasculation of mature flowers (dark yellow petal, and/or petal not opened) (Stage II); and 3) emasculation of mature flowers (dark yellow petal, and/or petal partially opened) (Stage III). Complete success of cross fertilization was for the interaction of Stage II (dark yellow petal, and/or petal not opened) flower emasculation with pollination of emasculated flower after 24 h. The lowest (1.8%) percent of success of cross fertilization was at stage I (light yellow petal) flower emasculation with pollination of emasculated flower within 12 h. The low range, 1.8–5.3% of success of cross fertilization was at flower stage III (dark yellow petal, and/or petal partially opened) emasculation with no pollination of emasculated flower, indicating occurrence of an accidental self-fertilization. Stage of flower development stage I (light yellow petal) and pollination of emasculated flowers after 48 h could give the optimum percent of success of cross fertilization and avoid accidental self-pollination.