In this paper, a series of anion exchange membranes (AEMs) for alkaline direct methanol fuel cells are prepared from novel quaternized block copolymers PS-b-PIN, composed of a hydrophobic polystyrene (PS) segment and a quaternized polyisoprene (PIN) segment bearing pendant quaternary ammonium groups attached to the polyisoprene backbone via alkyl spacers. The AEMs of PS-b-PIN are prepared via quaternization and alkilinization of the preformed films of the block copolymer precursors PS-b-PIBr with tunable compositions for morphology manipulation of the AEMs. The unique molecular structure and the developed methodology offer several important advantages including accurate control of the molecular architecture, avoidance of the use of toxic and ill-defined chloromethylation, as well as the absence of benzyltrimethylammonium groups for improved alkaline stability. The morphology–transport properties interplays are systematically investigated, showing the interconnectivity of the hydroxide conducting domains is critical to hydroxide conduction but less important for methanol permeation. Effective hydroxide transport is achieved in an AEM possessing PIN domains with 3-dimensional continuity, suggested by a high hydroxide conductivity of 2.5 × 10⁻² S cm⁻¹ and a moderate ion exchange capacity of 0.95 mmol g⁻¹. Relatively good long term chemical stability in strong basic environment is observed and the associated degradation mechanisms are discussed.