We present a crystallography chip enablingﾠin situﾠroom temperature crystallography at microfocus synchrotron beamlines andﾠX-rayﾠfree-electron laser (X-FEL) sources. Compared to otherﾠin situapproaches, we observe extremely low background and highﾠdiffractionﾠdata quality. The chip design is robust and allows fast and efficient loading of thousands of small crystals. The ability to load a large number ofﾠproteinﾠcrystals, at room temperature and with high efficiency, into prescribed positions enables high throughput automated serial crystallography with microfocus synchrotron beamlines. In addition, we demonstrate the application of this chip for femtosecond time-resolved serial crystallography at the Linac Coherent Light Source (LCLS, Menlo Park, California, USA). The chip concept enables multiple images to be acquired from each crystal, allowing differential detection of changes inﾠdiffractionﾠintensities in order to obtain high signal-to-noise and fully exploit the time resolution capabilities of XFELs.