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Half-sibling (HS) kinship testing is more difficult and complicated. This article describes a case where we sought to establish if three children with different mothers (one boy and two girls), shared a common father. The three children and their mother, as well as the suspected father's elder brother took part in the identification. To investigate the application of multiple genetic markers in HS kinship testing, autosomal STR, Y-STR, and X-STR was performed on blood samples from the individuals of interest C1 (female), M1 (mother of C1), C2 (male), M2 (mother of C2), C3 (female), M3 (mother of C3), and UC (elder brother of the suspected biological father). The 24 Y-STR haplotypes of C2 and UC were identical, confirming that they come from the same paternal line. Among the 27 X-STRs tested in C1 and C3, 18 of them had different paternal alleles, so C1 and C3 were excluded as half-sibling kinship.39 autosomal STR were calculated by ITO method, the HSI (or AI) of the pairs C1-C3, C2-C3, and C3-UC are <1, so HS kinship between C1 and C3, and between C2 and C3 can be excluded and avuncular kinship between C3 and UC can be excluded. The HSI (or AI) of the pairs C1-C2, C1-UC, and C2-UC are much >1, which may supports HS kinship between C1 and C2 and avuncular kinship between C1 and UC, and between C2 and UC.15 autosomal STR were calculated by discriminant function, DHS3 > DR3 between C1 and C2, and thus they could be HS. DHS3 < DR3 between C1 and C3, and between C2 and C3, so C1 and C3, and C2 and C3 may be unrelated individuals. Based on the above results, we can conclude that a HS relationship may be existed between C1 and C2. There may be a avuncular relationships between C1 and UC and between C2 and UC. HS relationships between C3 and C1 and between C3 and C2 as well as an avuncular relationship between C3 and UC were not supported by this study. The application of multiple genetic marker testing should be more regularly applied in the evaluation of HS and avuncular relationships. Increasing the number of related individuals tested when evaluating kinship relationships provides more data for better identification. [ABSTRACT FROM AUTHOR]