Upland Cotton (Gossypium hirsutum) is an economically important natural fiber crop. MicroRNAs (miRNAs) are ~22 nucleotide small RNAs regulating the expression of eukaryotic genes. Relatively few cotton miRNAs have been discovered to date, and the distribution and expression of cotton miRNAs in different tissues in response to environmental stimuli is unknown. We generated 20 small RNA libraries using next generation sequencing (NGS), including 4 Illumina leaf libraries (15,690,561 reads), 14 leaf (total 261,672,203 reads) and 2 root (total 43,094,310 reads) libraries generated using the ABI Soild platform. Three additional cotton small RNA libraries were downloaded from NCBI. Bioinformatic analysis was conducted using a modified version of the miRDeep program for discovery of plant miRNAs. A total 198 known mature miRNAs were discovered, including 198, 153 and 113 from cotton leaf, root and fiber respectively. Forty-five unique mature miRNAs originating from the leaf were found in this study, no root- or fiber-specific miRNAs were found. Using a collection of cotton ESTs, the USDA CottonDB library, and the sequenced D-genome of G. raimondii, 179 new miRNAs precursors (pre-miRNAs) encoding 99 unique mature miRNAs were identified. The single nucleotide variation between the pre-miRNAs from upland cotton (AD genome) and diploid cotton, G. raimondii (D-genome) encoding the same miRNAs was observed. One precursor identified using G. raimondii appears able to generate two distinct miR2950s from each of its arms, making this the first report suggesting arm switching in the biogenesis of plant miRNAs.