The RNA interference (RNAi) mechanism is a conserved system of nucleic acid manipulation, based on the interaction between small RNA guide molecules and Argonaute effector proteins. RNAi pathways are found in the vast majority of eukaryotes, and have diversified into a broad array of functions including gene regulation, antiviral immunity and transposable element (TE) suppression. Many of these functional innovations coincide with duplication of Argonaute genes, suggesting that gene duplication may be a key driving force in the diversification of RNAi. However, few studies have explicitly investigated Argonaute evolution after duplication. In this thesis, I focused on the impact of gene duplication on the evolution of Argonaute genes. Argonaute genes in different species exhibit a broad array of functions; however, most of our knowledge of Argonaute function in the arthropods is based on studies in D. melanogaster. To compare the rate of duplication and its evolutionary effect between different Argonaute subclades, I quantified gene turnover rates and evolutionary rate change in Argonaute genes from 86 Dipteran species (Chapter 2). I find that duplication rate varies widely between subclades and lineages, and that duplication drives an increase in evolutionary rate, suggesting that functional divergence after Argonaute duplication is prevalent throughout the Diptera. In the obscura group of Drosophila I identified a series of recent duplications of Argonaute2 (Ago2), which has antiviral and anti-TE functions in D. melanogaster. To quantify the extent of functional divergence between these paralogues, I measured the expression of paralogues from three species (D. subobscura, D. obscura and D. pseudoobscura), in different tissues and under viral challenge (Chapter 3). I find that the majority of Ago2 paralogues have specialised to a derived testis-specific role, potentially to suppress TE activity or meiotic drive. While CRISPR-Cas9 mediated knockout of these genes ultimately proved unsuccessful (Chapter 5), the selective importance of their derived function is suggested by its multiple independent origins. Functional novelty, as appears to have evolved in the obscura group Ago2 paralogues, is often driven by strong selection. To quantify the evolutionary rate and positive selection on these paralogues, I gathered intraspecies polymorphism data for all paralogues in D. subobscura, D. obscura and D. pseudoobscura, combining this with publicly-available population genomic data for D. pseudoobscura (Chapter 4). I find that the majority of paralogues in all species have extremely low diversity, indicative of recent selection, and identify recent selective sweeps on three paralogues in D. pseudoobscura. This suggests that the majority of Ago2 paralogues in the obscura group are evolving under strong positive selection. In this thesis I have aimed to quantify the effect of gene duplication on Argonaute evolution. I find that Argonaute genes duplicate frequently in some lineages, resulting in the evolution of derived functions that may be driven by positive selection. This suggests that functional diversification is prevalent in eukaryotic RNAi, and is likely to coincide with expansion of the Argonaute gene family.