Downsizing of highly-boosted spark-ignition (SI) engines is limited by pre-ignition, which may lead to extremely strong knocking and severe engine damage. Unfortunately, the concerning mechanisms are generally not yet fully understood, although several possible reasons have been suggested in previous research. The primary objective of the present paper is to investigate the influence of molecular bio-fuel structure on the locations of pre-ignition in a realistic, highly-charged SI engine at low speed by state-of-the-art optical measurements. The latter are conducted by using a high-sensitivity UV endoscope and an intensified high-speed camera.Two recently tested bio-fuels, namely tetrahydro-2-methylfuran (2-MTHF) and 2-methylfuran (2-MF), are investigated. Compared to conventional fuels, they have potential advantages in the well-to-tank balance. In addition, both neat ethanol and conventional gasoline are used as fuels.Although a spark plug with adapted heat range is used, the results show that pre-ignition starts both at the spark-plug ground electrode and also remote from the surfaces in different cycles for both furans and the standard gasoline. The pre-ignition tendency of these fuels decreases with increasing RON. By contrast, ethanol reveals a relatively high pre-ignition tendency, although its octane numbers (RON and MON) are particularly high, and the influence of the spark plug is vanishing. The different behavior of ethanol can be likely explained by its evaporation characteristics, which lead to reduced charge temperature, deteriorated mixture preparation, and enhanced fuel-oil interaction by liner impingement. Accordingly, pre-ignition appears also to be affected by mixture formation processes, in particular for ethanol.