The transfer of Wolbachia between hosts (transfection) is used to study the Wolbachia-host interaction and for applied strategies to affect harmful insect populations. Two common transfection techniques involve embryonic microinjection to transfer 1) Wolbachia-infected embryo cytoplasm and 2) embryo homogenate. Although both techniques have proven successful, their transfection efficiency previously has not been compared. Characterization of Wolbachia infection in early embryos demonstrates a higher concentration of Wolbachia in the posterior of Drosophila embryos. Therefore, transfection efficiency may be higher with cytoplasm microinjected from the posterior of donor embryos relative to anterior cytoplasm. Transfection efficiency using embryo homogenate will be affected by the buffer type. To address these questions, we have compared microinjection of D. simulans eggs using the cytoplasm transfer and embryo homogenization techniques. Comparisons included cytoplasm transferred using anterior and posterior cytoplasm from Wolbachia-infected donor embryos. Three different buffers were compared in microinjecting egg homogenate. Our results show that microinjection of embryos homogenized in SPG buffer yielded the highest transfection success and that Wolbachia from anterior and posterior embryo cytoplasm are equally competent for establishing infection (although survivorship of injected hosts differed). We discuss the results in relation to intra- and interspecific Wolbachia transfection and attempts to re-establish in vivo Wolbachia infections from infections in tissue culture.