ABSTRACTSynthetic genome evolution provides a dynamic approach for systematically and straightforwardly exploring evolutionary processes. Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) is an evolutionary system intrinsic to the synthetic yeast genome that can rapidly drive structural variations. Here, we detect over 260000 rearrangement events after the SCRaMbLEing of a yeast strain harboring 5.5 synthetic yeast chromosomes (synII, synIII, synV, circular synVI, synIXR and synX). Remarkably, we find that the rearrangement events exhibit a specific landscape of frequency. We further reveal that the landscape is shaped by the combined effects of chromatin accessibility and spatial contact probability. The rearrangements tend to occur in 3D spatially proximal and chromatin-accessible regions. The enormous numbers of rearrangements mediated by SCRaMbLE provide a driving force to potentiate directed genome evolution, and the investigation of the rearrangement landscape offers mechanistic insights into the dynamics of genome evolution.