Volatile compounds can be produced by fermentation from genetically engineered microorganisms. Escherichia coli strains are mainly used for isoprene production owing to their higher titers; however, this has thus far been confined to only strains BL21, BL21 (DE3), Rosetta, and BW25113. Here, we tested four groups of E. coli strains for improved isoprene production, including K‐12 (DH5α, BW25113, W3110, MG1655, XL1‐Blue, and JM109), B [Rosetta (DE3), BL21, and BL21 (DE3)], Crooks C, and Waksman W strains. The isoprene productivity of BL21 and MG1655 was remarkably higher than that of the others in 5‐L fermentation, and scale‐up fermentation (300 L) of BL21 was successfully performed. This system shows potential for biobased production of fuel and volatile compounds in industrial applications.