The objective of this study was to determine if chlorophyll fluorescence could be used as an indicator of anaerobic respiration in broccoli (Brassica oleracea L., Italica group) during modified-atmosphere packaging (MAP). Two types of packages were used, PD-941 bags, which provided optimum MAP conditions for broccoli (≈3 kPa O2 plus 5 kPa CO2), and PD-961EZ bags, which allowed the CO2 to accumulate (≈11 kPa CO2). After 28 days in MAP at 1 °C, the broccoli from both types of bag had similar appearances and weight losses. However, broccoli held in the PD-961EZ bags had developed slight to moderate alcoholic off-odors and had higher ethanol, acetaldehyde, and ethyl acetate content, as compared with broccoli in PD-941 bags. Chlorophyll fluorescence parameters (Fv/Fm, T1/2, Fmd, and ΦPSII) were lower for broccoli held in the PD-961EZ bags than in PD-941 bags, and these differences increased with storage duration. These results indicate that chlorophyll fluorescence is a reliable, rapid, nondestructive indicator of broccoli quality during MAP, and that it could be used to determine if broccoli has developed off-odors without opening the bag and disrupting the package atmosphere.