Defect structure of MgB2 bulk and ultrafine particles, synthesized by solid state reaction route, have been investigated mainly by the aid of X-band electron paramagnetic resonance spectrometer. Two different amorphous Boron (B) precursors were used for the synthesis of MgB2, namely, boron 95 (purity 95%–97%, <1.5 μm) and nanoboron (purity >98.5%, <250 nm), which revealed bulk and nanosized MgB2, respectively. Scanning and transmission electron microscopy analysis demonstrate uniform and ultrafine morphology for nanosized MgB2 in comparison with bulk MgB2. Powder X-ray diffraction data show that the concentration of the by-product MgO is significantly reduced when nanoboron is employed as precursor. It is observed that a significant average particle size reduction for MgB2 can be achieved only by using B particles of micron or nano size. The origin and the role of defect centers were also investigated and the results proved that at nanoscale MgB2 material contains Mg vacancies. Such vacancies influence the connectivity and the conductivity properties which are crucial for the superconductivity applications.