Success of discovery programs for microbial natural products is dependent on quick and concise discrimination between isolates from diverse environments. However, laboratory isolation and identification of priority genera using current 16S rRNA PCR-based methods are both challenging and time-consuming. An emerging strategy for rapid isolate discrimination is protein fingerprinting via matrix-assisted laser desorption ionization (MALDI) mass spectrometry. Using our in-house environmental isolate repository, we have created a main spectral (MSP) library for the Bruker Biotyper MALDI mass spectrometer that contains 95 entries, including , , and other environmentally related genera. The library creation required the acquisition of over 2,250 mass spectra, which were manually reviewed for quality control and consolidated into a single reference library using a commercial software platform. We tested the effectiveness of the reference library by analyzing 49 environmental isolate strains using two different sample preparation methods. Overall, this approach correctly identified all strains to the genus level provided that suitable reference spectra were present in the MSP library. In this study, we present a fast, accurate method for taxonomic assignment of environmentally derived bacteria from the order , providing a valuable alternative to traditional PCR-based methods. The MSP library described in the manuscript is available for use. The Gram-negative proteobacterial order has emerged as a promising source of novel natural products in recent years. This order includes the genus and the newly defined genera and However, development of this resource has been hampered by difficulties with rapid and selective isolation of strains from the environment. Environmental metagenome sequencing has revealed that the potential for natural products is not evenly distributed throughout the microbial world. Thus, large but targeted microbial isolate libraries are needed to effectively explore the chemical potential of natural products. To study these organisms efficiently, methods to quickly identify isolates to the genus level are required. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is already used in clinical settings to reliably identify unknown bacterial pathogens. We have adapted similar methodology using the MALDI Biotyper instrument to rapidly identify environmental isolates of , , and for downstream natural product discovery.