An increasing number of microRNAs (miRNAs) have been shown to play crucial regulatory roles in the process of plant development. Here, we used high-throughput sequencing combined with computational analysis to characterize miRNAomes from the ovules of wild-type upland cotton and a fiberless mutant during fiber initiation. Comparative miRNAome analysis combined with northern blotting and RACE-PCR revealed seven fiber initiation-related miRNAs expressed in cotton ovules and experimentally validated targets of these miRNAs are involved in different cellular responses and metabolic processes, including transcriptional regulation, auxin and gibberellin signal transduction, actin bundles, and lignin biosynthesis. This paper describes a complex regulatory network consisting of these miRNAs expressed in cotton ovules to coordinate fiber initiation responses. In addition, 36 novel miRNAs and two conserved miRNAs were newly identified, nearly doubling the number of known cotton miRNA families to a total of 78. Furthermore, a chromatin remodeling complex subunit and a pre-mRNA splicing factor are shown for the first time to be miRNA targets. To our knowledge, this study is the first systematic investigation of fiber initiation-related miRNAs and their targets in the developing cotton ovule, deepening our understanding of the important regulatory functions of miRNAs in cotton fiber initiation.