In order to systematically identify the critical genes involved in the response to low inorganic phosphorus (Pi) in cotton, we performed RNA sequencing (RNA-seq) analyses of root, stem, and leaf from Gossypium hirsutum seedlings grown under both Pi replete and Pi deplete conditions. In total, 1679, 1611, and 841 differentially expressed genes (DEGs) were observed in the roots, stems, and leaves, respectively. Several Pi-starvation-induced genes homologous to Arabidopsis were identified, such as SPX, PHO1/2, and PHF1, as well as several members of the PHT1 family, which showed different expression levels in specific organs. Subsequently, 25 randomly selected DEGs were validated by quantitative real-time PCR, the result of which was consistent with the RNA sequencing data, indicating the reliability of RNA-seq data. By analyzing the differentially expressed transcription factors, we found that the MYB family was one of the three most abundant transcription factor families in all three tissues. Moreover, some phytohormone-related DEGs were also found in G. hirsutum seedlings in this study. In particular, some auxin-responsive genes, such as IAA24, IAA11/14/16, and IAA4/14/16/29 were differentially expressed in the roots, stems and leaves of G. hirsutum seedlings respectively. Finally, homologous genes for some important phosphatases such as PPCK1 and VTC4 were also found to be differentially expressed. On analyzing the protein interaction network of 121 shared DEGs, five interaction networks - two of which contained 10 and 6 DEGs, respectively - were identified. These findings provided a theoretical basis for studying the gene function of different cotton tissues in response to low Pi stress.