Reproduction-manipulating bacteria like Wolbachia can shift sex ratios in insects towards females, but skewed sex ratios may also arise from genetic conflicts. The flea beetle Altica lythri harbors three main mtDNA strains that are coupled to three different Wolbachia infections. Depending on the mtDNA types, the females produce either offspring with a balanced sex ratio or exclusively daughters. To obtain markers that can monitor when sex bias arises in the beetle’s ontogeny, we elucidated the sex determination cascade of A. lythri. We established a RT-PCR method based on length variants of dsx (doublesex) transcripts to determine the sex of morphologically indistinguishable eggs and larvae. In females of one mtDNA type (HT1/HT1*) known to produce only daughters, male offspring were already missing at the egg stage while for females of another type (HT2), the dsx splice variants revealed a balanced sex ratio among eggs and larvae. Our data suggest that the sex determination cascade in A. lythri is initiated by maternally transmitted female-specific tra (transformer) mRNA as primary signal. This tra mRNA seems to be involved in a positive feedback loop that maintains the production of the female splice variant, as known for female offspring in Tribolium castaneum. The translation of the maternally transmitted female tra mRNA must be inhibited in male offspring, but the underlying primary genetic signal remains to be identified. We discuss which differences between the mtDNA types can influence sex determination and lead to the skewed sex ratio of HT1.