Maskless photolithography, a useful tool used in patterning the photoresist which acts as a mask prior to the actual etching process of substrate, has attracted attention mainly due to the taking advantage of reducing cost because of not requiring a preprepared mask and freedom in creating the desired pattern on any kind of substrate. In this study, we performed the positive photoresist patterning with microstructures on both glass and silicon substrates via maskless photolithography. Specifically, we examined the discrepancies between the transparent (glass) and reflective (silicon) substrates even though the photolithographic process has been carried out under the same conditions. Since the positive photoresist patterning was the subject of this study, we could successfully produce the microholes with almost circular shapes and properly placed in squarely packed on both substrates as confirmed by optical microscopy and profilometer mapping measurements. We observed additional rings around the holes when silicon was used as substrate while very clear microholes were obtained for glass. Besides, the number of the rings increased when the writing speed of laser (velocity) reduced. We claim that these important findings can be attributed to the standing wave effect phenomenon which results from the multiple reflections through the semi-transparent photoresist coated on the reflective surface of the polished silicon. In brief, we reveal an important conclusion, in this study, based on the differences in formation of the microholes only due to the substate preference while all the photolithographic process parameters are kept the same.