Somatic embryogenesis (SE) is a critical transition from vegetative to embryogenic growth in higher plants; however, few studies have investigated the mechanism that regulates SE initial differentiation. Most cotton varieties have not undergone regeneration by SE, so only a few varieties can be used in genetic engineering. Here, two varieties of cotton with different SE capabilities (HD, higher differentiation and LD, lower differentiation) were analyzed by high throughout RNA-Seq at the pre-induction stage (0h) and two induction stages (3h and 3d) under callus-induction medium (CIM). About 1150 million clean reads were obtained from 98.21% raw data. Transcriptomic analysis revealed that "protein kinase activity" and "oxidoreductase activity" were highly represented GO terms during the same and different treatment stages among HD and LD. Moreover, several stress-related transcription factors might play important roles in SE initiation. The SE-related regulation genes (SERKs) showed different expression patterns between HD and LD. Furthermore, the complex auxin and ethylene signaling pathway contributes to initiation of differentiation in SE. Thus, our RNA-sequencing of comparative transcriptome analysis will lay a foundation for future studies to better define early somatic formation in cotton with different SE capabilities.