The positive transcription elongation factor (P‐TEFb; CDK9/cyclin T1) regulates RNA polymerase II‐dependent transcription of cellular and integrated viral genes. It is an essential cofactor for HIV‐1 Tat transactivation, and selective inhibition of P‐TEFb blocks HIV‐1 replication without affecting cellular transcription; this indicates that P‐TEFb could be a potential target for developing anti‐HIV‐1 therapeutics. Flavopiridol, a small molecule CDK inhibitor, blocks HIV‐1 Tat transactivation and viral replication by inhibiting P‐TEFb kinase activity, but it is highly cytotoxic. In the search for selective and less cytotoxic P‐TEFb inhibitors, we prepared a series of flavopiridol analogues and evaluated their kinase inhibitory activity against P‐TEFb and CDK2/cyclin A, and tested their cellular antiviral potency and cytotoxicity. We identified several analogues that selectively inhibit P‐TEFb kinase activity in vitro and show antiviral potency comparable to that of flavopiridol, but with significantly reduced cytotoxicity. These compounds are valuable molecular probes for understanding P‐TEFb‐regulated cellular and HIV‐1 gene transcription and provide potential anti‐HIV‐1 therapeutics.