Somatic embryogenesis (SE) plays a vital role in genetic transformation and massive propagation of important agronomical and economical crops.  Here, we conducted a systematic assessment of the morphological, cytochemical, and cytogenetical characteristics of six culture strains with various embryogenic/regenerative potential during SE process in cotton.  Results indicated that the six cell culture strains had stable ploidy levels, and did not reveal any relationship between the cytogenetic state and their morphogenetic potential.  Moreover, the six culture strains were compared via double staining with Evans blue and Acetocarmine to efficiently distinguish embryogenic and non-embryogenic cells and determine the embryogenic nature of the calli.  In addition, the kind of auxins added in medium affected not only growth property, color, size of cell clumps but also ploidy level and regeneration ability.  By combining analysis of morphological, cytochemical, and cytogenetical characteristics of the cell cultures, we are able to obtain and maintain homogeneous cell population with high morphogenic and regeneration ability and establish efficient somatic embryogenesis and regeneration system from short-term cell cultures in upland cotton, which highlight the application of biotechnological approaches in crop breeding, and above all, to better understand totipotency of cells in higher plants.