The aggregation of alpha-synuclein (alpha-SYN) into fibrils is characteristic for several neurodegenerative diseases, including Parkinson's disease (PD). Ninety percent of alpha-SYN deposited in Lewy Bodies, a pathological hallmark of PD, is phosphorylated on serine129. alpha-SYN can also be phosphorylated on tyrosine125, which is believed to regulate the membrane binding capacity and thus possibly its normal function. A better understanding of the effect of phosphorylation on the aggregation of alpha-SYN might shed light on its role in the pathogenesis of PD. In this study we compare the aggregation properties of WT alpha-SYN with the phospho-dead and phospho-mimic mutants S129A, S129D, Y125F and Y125E and in vitro phosphorylated alpha-SYN using turbidity, thioflavin T and circular dichroism measurements as well as transmission electron microscopy. We show that the mutants S129A and S129D behave similarly compared to wild type (WT) alpha-SYN, while the mutants Y125F and Y125E fibrillate significantly slower, although all mutants form fibrillar structures similar to the WT protein. In contrast, in vitro phosphorylation of alpha-SYN on either S129 or Y125 does not significantly affect the fibrillization kinetics. Moreover, FK506 binding proteins (FKBPs), enzymes with peptidyl-prolyl cis-trans isomerase activity, still accelerate the aggregation of phosphorylated alpha-SYN in vitro, as was shown previously for WT alpha-SYN. In conclusion, our results illustrate that phosphorylation mutants can display different aggregation properties compared to the more biologically relevant phosphorylated form of alpha-SYN.