Between January 2001 and May 2003, 167 stream segments on the islands of Kaua‘i, O‘ahu, Maui, and Hawai‘i were sampled for stream macroalgae and measured for a series of physical and chemical conditions. Conditions ranged more widely than previously reported, which is likely due to the greater diversity of habitats accessed and the year-round sampling representation in this study. Water temperature ranged from 12.5 to 27.5 °C (mean = 21.4 °C ± 2.4), pH from 5.5 to 8.9 (mean = 7.8 ± 0.5), and specific conductance from 20 to 490 μS·cm⁻¹ (mean = 102 μS·cm⁻¹ ± 75.9). A total of 160 specific and subspecific taxa was identified, of which 27 are new records for the Hawaiian Archipelago. The Chlorophyta compose the majority of the taxa, followed by the Cyanobacteria, Rhodophyta, Bacillariophyta, and Tribophyta. The mean number of taxa per stream segment was 5.0 ± 2.7, which is the highest such value reported. Grouping of taxa by morphological form demonstrates that the majority of taxa were free filaments (58%), followed by mats (17%), tufts (13%), and gelatinous colonies (9%). A principal coordinates analysis of the stream sites indicated that a high degree of overlap in floristic composition is evident for most of the Islands, and only sites on the island of Hawai‘i exhibit a localized positioning to one side of the principal coordinates biplot. The flora of Hawai‘i Island appears to be unique only in the sense that it contains fewer broadly distributed taxa than the remaining islands, which may be a function of island age. Cluster analysis of the islands based on two types of comparisons suggests stronger similarities between the islands of Maui and Kaua‘i, and O‘ahu and Hawai‘i than previously reported. The Hawaiian stream macroalgal flora contains a number of cosmopolitan taxa, although it is recognized that concepts of some of these taxa may change with additional data.