Periodic π-vacancies in graphene superlattices (GSLs) provide a symmetry-based route to band gap opening in graphene by modifying the π-band dispersion, yet the symmetry conditions that determine whether a vacancy motif can open a band gap remain unclear. Here, we investigate periodic π-vacancy GSLs using a nearest-neighbor tight-binding model with one orbital per carbon site to identify the symmetry requirements for band gap opening. Here, π vacancies, resembling functionalized, substituted, or missing carbon sites, are modeled as site deletions in the π basis, and the corresponding hopping matrix elements to and from the deleted sites are set to zero. We focus on π-vacancy motifs with and point groups. A GSL, where is the integer scaling factor multiplying the honeycomb primitive-cell vectors, is required to fold and to , which is one of the requirements for opening a band gap. For -type vacancies, the Dirac cones are pinned at the high-symmetry points, thus remaining at in the folded GSLs. However, those -type vacancies, which reduce the global point group of the GSL to by preserving a pair of perpendicular mirror symmetries (, can constrain the positions of Dirac cones at . The absence of the and planes allows the cones to shift around in the superlattice. The -type vacancies open a relatively larger band gap than the -type vacancies due to twofold degeneracy at , while the -type vacancies leave the GSL with nondegenerate bands. The energy contours near the Dirac cone for -symmetric vacancies remain isotropic, while the absence of generates anisotropic cones. The band gap openings in superlattices scale linearly with defect concentration for each vacancy motif, with the motif yielding the largest gap, reaching 314 meV at only 3.7% defect concentration. Disorder of the superlattice by missing vacancies or by vacancy displacements of one or two primitive lattice vectors of graphene breaks the symmetry, lifts the degeneracy, and reduces the gap toward the pristine graphene limit.