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The cytochrome P450 (CYP450) monooxygenase superfamily, which is involved in the biosynthesis pathways of many primary and secondary metabolites, plays prominent roles in plant growth and development. However, systemic information about CYP450s in <i>Brassica napus</i> (BnCYP450) was previously undiscovered and their biological significance are far from understood. Members of clan 86 CYP450s, such as CYP704Bs, are essential for the formation of pollen exine in plant male reproduction, and the targeted mutagenesis of <i>CYP704B</i> genes has been used to create new male sterile lines in many crops. In the present study, a total of 687 <i>BnCYP450</i> genes were identified in <i>Brassica napus</i> cultivar “Zhongshuang 11” (ZS11), which has nearly 2.8-fold as many <i>CYP450</i> members as in <i>Arabidopsis thaliana</i>. It is rationally estimated since <i>Brassica napus</i> is a tetraploid oil plant with a larger genome compared with <i>Arabidopsis thaliana</i>. The <i>BnCYP450</i> genes were divided into 47 subfamilies and clustered into nine clans. Phylogenetic relationship analysis reveals that <i>CYP86</i> clan consists of four subfamilies and 109 <i>BnCYP450s</i>. Members of <i>CYP86</i> clan genes display specific expression profiles in different tissues and in response to ABA and abiotic stresses. Two <i>BnCYP450s</i> within the <i>CYP704</i> subfamily from <i>CYP86</i> clan, <i>BnCYP704B1a</i> and <i>BnCYP704B1b</i>, display high similarity to <i>MS26</i> (<i>Male Sterility 26</i>, also known as <i>CYP704B1</i>). These two <i>BnCYP704B1</i> genes were specifically expressed in young buds. We then simultaneously knocked-out these two <i>BnCYP704B1</i> genes through a clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) genome engineering system. The edited plants displayed a pollenless, sterile phenotype in mature anthers, suggesting that we successfully reproduced genic male sterility (GMS, also known as nuclear male sterility) lines in <i>Brassica napus</i>. This study provides a systemic view of <i>BnCYP450s</i> and offers a strategy to facilitate the commercial utility of the CRISPR/Cas9 system for the rapid generation of GMS in rapeseed via knocking-out <i>GMS</i> controlling genes.