We previously discovered a positive allosteric modulator (PAM) of the metabotropic glutamate receptor subtype 5 (mGlu5) termed 4 N- 4-chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl]phenyl -2-hydroxybenzamide (CPPHA) that elicits receptor activation through a novel allosteric site on mGlu5, distinct from the classical mGlu5 negative allosteric modulator (NAM) MPEP allosteric site. However, a shallow structurea??activity relationship (SAR), poor physiochemical properties, and weak PAM activity at rat mGlu5 limited the utility of CPPHA to explore allosteric activation of mGlu5 at a non-MPEP site. Thus, we performed a functional high-throughput screen (HTS) and identified a novel mGlu5 PAM benzamide scaffold, exemplified by VU0001850 (EC50 = 1.3 I?M, 106 Glumax) and VU0040237 (EC50 = 350 nM, 84 Glu Max). An iterative parallel synthesis approach delivered 22 analogues, optimized mGlu5 PAM activity to afford VU0357121 (EC50 = 33 nM, 92 Glumax), and also revealed the first non-MPEP site neutral allosteric ligand (VU0365396). Like CPPHA, PAMs within this class do not appear to bind at the MPEP allosteric site based on radioligand binding studies. Moreover, mutagenesis studies indicate that VU0357121 and related analogues bind to a yet uncharacterized allosteric site on mGlu5, distinct from CPPHA, yet share a functional interaction with the MPEP site.