Self-assembled monolayers (SAMs) of ω-(4′-methylbiphenyl-4-yl)ethanethiol (CH3(C6H4)2(CH2)2SH, BP2) prepared at different temperatures on Au(111) substrates were investigated using scanning tunneling microscopy (STM). Also, the effect of the incubation time of the gold substrate in the thiol solution was examined. The STM results showed that samples prepared at room temperature were significantly different from those prepared at elevated temperatures in their surface morphology, space group and size of unit cell. The micrographs of samples prepared at higher temperatures revealed a pronounced and progressive increase in the size of the well-known etch-pits at the expense of their density with increasing preparation temperature (but the increase did not continue for SAMs prepared at 348 K). The average domain size was found to increase significantly with increasing preparation temperature. In addition, polymorphism was observed in BP2 SAMs at all investigated temperatures. This study has demonstrated that solution temperature and incubation time are key factors controlling the two-dimensional SAM structure of BP2 molecules.