Abstract: To study the regulation and formation mechanism of premature flowering and premature flowering resistance of flue-cured tobacco, we used the Agilent Tobacco Oligo Microarray to deduce the expression profiling of flue-cured tobacco K326 and its premature flowering resistance variety Huayan06 under the flower bud differentiation process. The result showed that the same days from their flower bud differentiation the number of differential probes were the maximum amount of the comparative item between K326 and Huayan06. The number of the probe was 5295. On the day of K326 flower bud differentiation, the number of differential probe was the minimum amount. The number of the probe was 2116. The molecular function mainly included catalytic activity, transporter activity, binding and transcription regulator activity. The cellular component mainly included cell, cell part and organelle. The biological process was the most complex, involved plant resistance, growth, development, etc. It would establish a basis for the further study and search for genes associated with tobacco flowering.