This repository contains focus-filtered timelapse microscopy data of two interfertile Chlamydomonas algal species. The protocol to generate this data is described in the associated publication, "Phenotypic differences between interfertile Chlamydomonas species", and summarized here. Cells were collected from agar plates and suspended in water, then left to sit overnight to encourage gamete formation. During this time, non-motile cells settled, allowing for the enrichment of motile cells in the supernatant. These enriched cells were then loaded onto agar microchambers (100 micron diameter and 40 micron depth) for imaging. We collected videos on a Nikon Ti2-E microscope equipped with a Photometrics Kinetix digital scMos camera. We performed differential interference contrast (DIC) imaging using a Plan Apo 10× 0.45 Air objective. We collected videos with a 5.1 ms exposure with acquisition every 50 ms for three minutes. We placed a red light filter [IR longpass, 610 nm (ThorLabs)] in the light path to maintain swimming behavior of cells. The procedure was standardized and repeated four times to ensure consistency. Focus-filtered timelapse data of C. reinhardtii or C. smithii cells in agar microchamber wells are shared here. The code for focus-filtering and collection of measurements can be found in the associated Github repository. Reference Essock-Burns T, Garcia III G, MacQuarrie CD, Mets DG, York R. (2023). Phenotypic differences between interfertile Chlamydomonas species Notes In addition to the raw data, the dataset includes sample images that are intermediates in the image processing pipeline, as well as 2D morphology measurements of the cells in a csv file. "Cr" indicates Chlamydomonas reinhardtii "Cs" indicates Chlamydomonas smithii Frame rate: 20 frames per second (fps) Pixel size: 0.6398 microns/pixel