Abstract: The cytological characteristics of broccoli (Brassica oleracea var. italica) microspore development, bud morphology in different microspore development periods and anther color were observed to identify the period of microspore development directly from the morphological characteristics of buds and anther of broccoli, which would provide the scientific evidence for culturing of anther and isolated microspore. The results showed that broccoli microspore development was divided to four stages: tetrad period, early or mid-uninucleate period, late-uninucleate period, and binuclear period, and each stage had obvious characteristics. The microspore development period of broccoli was closely related with bud size and anther color. The buds were 10.52-11.05 mm in longitudinal diameter and 3.64-4.25 mm in transversal diameter, and the anthers 2.85-2.87 mm in length and 0.95-0.96 mm in width. The petals were calyx slightly and the anthers and the petals were yellowish. Eighty percent microspores could develop to the stage of late-uninucleate.