Single celled fibers initiate at anthesis from cotton seed epidermal cells of normal developmental cotton cultivars; however, fiber initiation is retarded in some cotton fiber mutants. In this study, the relationship between genes associated with fiber initiation retardation and fiber initiation development was investigated using three cotton fiber developmental mutants: recessive naked seed n2; dominant naked seed N1; and Xinxiangxiaoji Linted Fuzzless Mutant (XinFLM); with genetic standard line TM-1 (TM-1) as control. Retardation during fiber initiation development was observed in N1 and XinFLM by scanning electron microscope (SEM) analysis. Reverse transcription polymerase chain reaction (RT PCR) analysis of genes related to the fiber initiation development showed that the expression of GhEXP1 and GhMYB25 was lower in N1 and XinFLM than in TM-1 and n2, however, the expression of GhTFG1 and GhTFG3 in XinFLM and n2 was higher than in TM-1 and N1. In rive and in vitro treatments on ovules demonstrated that 30% hydrogen peroxide (H202) could prevent fiber initiation retardation in XinFLM, but no evident effect on N1. To further confirm the relationship between gene expression and the effects of H2O2 in XinFLM, qRT PCR analysis of four differentially expressed genes was performed using-1 d post anthesis (DPA) ovules of XinFLM treated for 24 and 48h with 30% H2O2 and H2O, respectively, with 0 and 1 DPA untreated ovules fiom XinFLM and TM-1 as control. The results showed that the expression of GhMYB25 and GhEXP1 showed significant difference in XinFLM after-1 DPA ovule treated for 24h relative to the untreated or H2O treated ovules, with the expression of GhMYB25 increased significantly and that of GhEXP1 decreased. This implied that H2O2 might be one of the upstream signal molecules affecting the expression of GhMYB25 and GhEXP1 genes. The fiber initiation retardation in XinFLM might be related to the production of reactive oxygen species (ROS).