Fusarium oxysporum f. sp. vasinfectum race 4 (FOV4) is a soil-borne fungal pathogen threatening US cotton production. The objective of this study was to develop a reliable and efficient method to evaluate cotton for FOV4 resistance based on taproot rot during seed germination through five growth chamber tests and two greenhouse tests. Seeds from eight cotton cultivars (Set 1) were germinated in a paper towel for 6 days, and taproots were inoculated with a FOV4 conidial suspension using three inoculation methods, i.e., taproot dipping, taproot wounding, and paper towel drenching, in addition to seed soaking before germination. Taproot rot-based disease incidence (DI) and disease severity rating (DSR), seed germination percentage (SGP), and plant fresh weight (PFW) were measured 7 days after inoculation. Taproot dipping was the most efficient and reliable evaluation method. SGP and PFW were not significantly correlated with DI and DSR, making them inappropriate to use in resistance evaluation. Pima DP 359 RF and PHY 881 RF were the most resistant with the lowest root rot. The taproot dipping method was repeated in another test and confirmed in two tests using another set of eight cultivars (Set 2). The taproot rot-based DSR at germination was significantly correlated with DSR at the seedling stage in the greenhouse in both sets and with previous results in seedling mortality in the greenhouse and field in Set 2. The results suggest that the response to FOV4 infections at the seed germination stage is overall congruent with that at the seedling stage.