The manipulation and assembly of polystyrene-based Janus particles of varied surface chemistry on one hemispherical particle surface under high frequency nonuniform ac-electric fields is examined experimentally by in situ microscopic observation. Strong effects of ac-field frequency, medium conductivity, and particle surface chemistry on the structure of Janus colloidal assembly are observed. At low medium conductivity, σm from 0.0007 S/m to 0.0153 S/m, pearl chains of Janus particles are observed over the ac-frequency range from 25 kHz to 20 MHz, indicating the dielectrophoresis (DEP)-directed assembly. In contrast, the chaining of Janus particles is disrupted in a certain frequency range at high σm from 0.0153 S/m to 0.116 S/m, suggesting the combining effects of both induced-charge electrophoresis (ICEP) and DEP. The critical transition frequency for the onset of the fractal aggregation at high σm from 0.0153 S/m to 0.116 S/m is experimentally determined, showing a good agreement with the theoretically predicted upper ICEP frequency limit. Additionally, it is demonstrated that by using zwitterionic Janus particles, the assembled structure of Janus particles under ac-fields can be modified by the chemical coatings on each hemispherical surface of Janus particles.