Background: MicroRNAs (miRNAs) are small, endogenously expressed, non-coding RNA molecules involved in gene transcription and expression that combine with specific mRNA site of target genes to inhibit protein synthesis or degrade mRNA. Since the first plant miRNA was reported in 2002, numerous new miRNAs and their targets have been discovered via high-throughput sequencing and computational approaches. However, the genetic variation of miRNA genes is poorly understood due to the lack of miRNA-specific DNA markers. Results: To study the genetic variation and map miRNAs and their putative target genes in cotton, we designed specific primers based on pre-miRNAs and published putative target genes. A total of 83 pre-miRNA primers and 1,255 putative target gene primers were surveyed, and 9 pre-miRNA polymorphic loci were mapped on 7 of the 26 tetraploid cotton chromosomes. Furthermore, 156 polymorphic loci of the target genes were mapped on the cotton genome. To map more miRNA loci, miRNA-based SRAP (sequence-related amplified polymorphism) markers were used to map an additional 54 polymorphic loci on the cotton genome with the exception of Chr01, Chr22, and Chr24. Finally, a network between miRNAs and their targets was constructed. All pre-miRNAs and 98 putative target genes were selected for RT-PCR analysis, revealing unique expression patterns across different fiber development stages between the mapping parents. Conclusions: Our data provide an overview of miRNAs, their putative targets, and their network in cotton as well as comparative expression analyses between Gossypium hirsutum and G. barbadense. These data provide a foundation for understanding miRNA regulation during cotton fiber development.