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Improving the quality of Australian cotton fiber is essential for maintaining industry viability. Two field experiments were conducted to assess the fiber quality and yarn performance of Australian bred cotton (five Gossypium hirsutum L. and one G. barbadense L.) genotypes. The work included the novel measurement of fiber maturity ratio, fiber linear density, and fiber diameter (ribbon width). The strongest yarns were produced using genotypes with the longest and finest fiber, for example, the strength of 20 tex yarns for the G. barbadense L. cultivar Sipima 280 (length = 36.6 mm, linear density = 143 mtex, ribbon width = 13.7 μm) was 25.4 cN tex−1 cf. the G. hirsutum L. cultivar Sicala 350B (length = 32.5 mm, linear density = 185 mtex, ribbon width = 14.5 μm) yarn strength of 18.1 cN tex−1 Micronaire was an inferior indicator of yarn performance, for example, the G. hirsutum L. breeding lines CHQX12B and CHQX377 each had micronaire values of 4.4, but CHQX377 spun stronger yarns due to its finer and more mature fiber. Lint cleaning had the greatest influence on nep (fiber knot) generation for G. hirsutum L. genotypes, generating on average 104 neps g−1 per lint cleaner passage. There was a negative association between fiber quality and yield, and a cost benefit analysis showed that fiber yield was the dominant economic factor compared to price premiums for better fiber quality. Alternative methods of determining fiber fineness will improve the value of Australian cotton.